Methods for treating autism spectrum disorder and associated symptoms

ABSTRACT

The present disclosure relates to compositions and methods for treating autism spectrum disorder (ASD) by restoring an ASD patient&#39;s gut microbiota. These methods can be used with ASD patient with or without ongoing gastrointestinal symptoms. Provided here is a method for ASD treatment in a subject in need thereof comprising or consisting essentially of administering a therapeutic composition comprising a fecal microbe or a fecal microbiota preparation to the subject. Also provided here is a method comprises administering an antibiotic to a human subject; subjecting the human subject to a bowel cleanse; and administering purified fecal microbiota to the human subject. Further provided are evaluation and quantitative characterization of patient symptom improvements upon treatment described here.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional PatentApplication Nos. 62/165,556, filed on May 22, 2015, which isincorporated herein by reference in its entirety.

BACKGROUND

The present disclosure relates to methods of treating autism spectrumdisorder (ASD). Autism spectrum disorder (ASD) is a complexneurodevelopmental condition characterized by widespread abnormalitiesof social interactions and communication, as well as restrictedinterests and repetitive behaviors. ASD typically appears during thefirst three years of life and manifests in characteristic symptoms orbehavioral traits. A diagnosis of ASD now includes several conditionsthat used to be diagnosed separately: autistic disorder, pervasivedevelopmental disorder not otherwise specified (PDD-NOS), and Aspergersyndrome. All of these conditions are now encompassed by the diagnosticcriteria for autism spectrum disorder as set forth in the AmericanPsychiatric Association's Diagnostic & Statistical Manual of MentalDisorders, Fifth Edition (DSM-V).

In addition to the spectrum of symptoms seen within these principaldiagnostic criteria, ASD individuals display a wide range ofneurological comorbidities, including intellectual disability, epilepsy,and anxiety and mood disorders, as well as non-neurologicalcomorbidities, including blood hyperserotonemia, immune dysregulation,and GI dysfunction (e.g., chronic constipation, diarrhea, abdominalpain, and gastroesophageal reflux).

To date, there are no FDA-approved treatments for reducing oreliminating the core symptoms of autism spectrum disorder. The only twomedications approved by the FDA for treating autism, risperidone (soldunder Risperdal®) and aripiprazole (sold under Abilify®), arespecifically indicated for reducing irritability in subjects having ASD.Accordingly, there remains a need in the art for improved methods fortreating and reducing the severity and incidence of symptoms associatedwith autism spectrum disorder. This application provides a method fortreating an ASD patient (with or without a GI symptom) by transferringbeneficial fecal bacteria to replace, restore, or rebalance the ASDpatient's gut microbiota, a treatment referred to here as MicrobiotaTransfer Therapy (MTT).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents Gastrointestinal Symptom Rating Scale (GSRS) data fortrial participants.

FIG. 2 presents GSRS subscale data collected prior to (“pre”) andfollowing (“post”) MTT treatment.

FIG. 3 presents continuous improvements of both average GSRS and averagePGI-R scores of the participants.

FIG. 4 presents Aberrant Behavior Checklist (ABC) data for MTT trialparticipants.

FIG. 5 presents GSRS scores collected 8 weeks post-treatment.

FIG. 6 is a graph demonstrating the lack of correlation between age andthe degree of CARS score improvement.

FIG. 7 is a graph demonstrating that end-of-treatment PGI-R scores hadlittle correlation with age.

FIG. 8 is a graph demonstrating the lack of correlation between thedegree of improvement on CARS and initial GSRS score.

FIG. 9 (including panels a to e) describes the improvement of GI- andcore ASD-related symptoms of 18 ASD-afflicted children treated with MTT.Children were treated with vancomycin for two weeks followed by theadministering of a fecal bacteria composition for 8 weeks, with a singlefollow-up evaluation 8 weeks after treatment ended. Panel a, Changes inGSRS scores. GSRS is scored on a Likert scale from 1 (no symptoms) to 7(very severe discomfort). Panel b, Changes in PGI-R scores (Overallautism/related symptoms). PGI-R is scored from −3 (much worse), −2(worse), −1 (slightly worse), 0 (no change), 1 (slightly better), 2(better) to 3 (much better) compared to baseline. Panel c, CARSassessment pre-treatment, post treatment and 8 weeks post treatment.Panel d, Total SRS score pre-treatment, post treatment and 8 weeks posttreatment. Panel e, Total ABC score pre-treatment, post treatment and 8weeks post treatment. The data points represent 18 individualparticipants, and some data points overlap in the box plot. Asterisks(at the top of the box-plot) indicate whether individuals (at each timepoints) have significantly decreased since pre-treatment (Week 0). ns:not-significant, *: p<0.05, **: p<0.01, ***: p<0.001 (two-tailed pairedt-test). Two participants who had less than 50% improvement in GSRSscores are defined as non-responders and color-coded in grey.

FIG. 10 (including panels a to c) provides a breakdown of GSRScomponents and improvements in patients. a, GSRS subscores at baseline,MTT treatment end, and 8 weeks after treatment. b, Results of dailystool records, averaged over 2 weeks. c, Subscales of the ABC vs. time.*: p<0.05, **: p<0.01, ***: p<0.001 (two-tailed paired t-test).

FIG. 11 demonstrates a correlation between GSRS and PGI-R (based on thedata shown in FIG. 10, panels a and b). The Pearson correlation testshowed r=−0.56 and p<0.001.

FIG. 12 shows Vineland Developmental Age (in years) for differentsubscales and for the average of all the subscales, measured at baselineand at the end of observation 4 months later. Note that the averagechronological age was 10.9 years at the start of treatment, so atbaseline there were delays in all areas, especially in the core autismareas of language and social (interpersonal) ability. *: p<0.05, **:p<0.01, ***: p<0.001 (two-tailed paired t-test).

FIG. 13 shows subscores of the PGI-R at end of treatment (week 10). Thescale goes from 3 (much better) to 2 (better) to 1 (slightly better) to0 (no change) to minus 3 (much worse). Scores were similar after 8 weeksof no treatment (week 18). The data points represent 18 individualparticipants, and some data points overlap in the box plot.

SUMMARY

This application provides a method for treating an autism spectrumdisorder (ASD) in a subject in need thereof, the method comprisingadministering to the subject an amount of a pharmaceutical compositioneffective for treating the ASD, where the pharmaceutical compositioncomprises a fecal microbe preparation, where the subject exhibits atleast a 10% reduction in ASD symptom severity after the treatment ascompared to before initiating the treatment, and based on an assessmentsystem selected from the group consisting of Childhood Autism RatingScale (CARS), Childhood Autism Rating Scale 2-Standard Form (CARS2-ST),Childhood Autism Rating Scale 2-High Functioning (CARS2-HF), AberrantBehavior Checklist (ABC), Social Responsiveness Scale (SRS), andVineland Adaptive Behavior Scale II (VABS-II).

Also provided here is a method for reducing autism severity in anautistic human subject comprising or consisting essentially of thefollowing steps: administering an antibiotic to an autistic humansubject; subjecting the autistic human subject to a bowel cleanse; andadministering a fecal microbiota preparation to the human subject,wherein the human subject exhibits a significant reduction in autismsymptom severity after said method as compared to before initiating themethod.

Further provided here is a method for treating an autism spectrumdisorder (ASD) in a human subject in need thereof, where the methodcomprises or consists essentially of administering a therapeuticcomposition comprising fecal microbes or a fecal microbiota to the humansubject, wherein the human subject exhibits at least a 10% or 20%reduction in autism symptom severity as assessed by CARS, CARS2-ST,CARS2-HF, ABC, SRS, or VABS-II relative to severity as assessed prior toinitiating the treatment, wherein the human subject further exhibits agastrointestinal (GI) symptom and the GI symptom severity is reduced byat least 40% as assessed by Gastrointestinal Symptom Rating Scale (GSRS)relative to severity as assessed prior to initiating the treatment.

Also provided here is a method for treating an autism spectrum disorder(ASD) in a human subject in need thereof, where the method comprises orconsists essentially of the following steps: orally-administering anantibiotic to the human subject; subjecting the human subject to a bowelcleanse; and administering a therapeutic composition comprising fecalmicrobes or a fecal microbiota to the human subject, wherein the humansubject exhibits at least a 10% or 20% reduction in autism symptomseverity as assessed by CARS, CARS2-ST, CARS2-HF, ABC, SRS, or VABS-IIrelative to severity as assessed prior to initiating the treatment,wherein the human subject further exhibits a gastrointestinal (GI)symptom and the GI symptom severity is reduced by at least 40% asassessed by GSRS relative to severity as assessed prior to initiatingthe treatment.

In another aspect, the present disclosure also discloses the use of afecal microbe or a fecal microbiota preparation in the manufacture of amedicament for the treatment of an autism spectrum disorder (ASD) in asubject in need thereof, where the subject exhibits at least a 10%reduction in ASD symptom severity after the treatment as compared tobefore initiating the treatment, and based on an assessment systemselected from the group consisting of Childhood Autism Rating Scale(CARS), Childhood Autism Rating Scale 2-Standard Form (CARS2-ST),Childhood Autism Rating Scale 2-High Functioning (CARS2-HF), AberrantBehavior Checklist (ABC), Social Responsiveness Scale (SRS), andVineland Adaptive Behavior Scale II (VABS-II).

The present disclosure also discloses the use of a fecal microbe or afecal microbiota preparation in the treatment of an autism spectrumdisorder (ASD) in a human subject in need thereof, wherein the humansubject further exhibits a gastrointestinal (GI) symptom, where thetreatment comprises or consists essentially of: administering anantibiotic to the human subject; subjecting the human subject to a bowelcleanse; and administering a therapeutic composition comprising fecalmicrobes or a fecal microbiota to the human subject, wherein the humansubject exhibits at least a 10% or 20% reduction after a less than12-week treatment in autism symptom severity as assessed by CARS,CARS2-ST, CARS2-HF, ABC, SRS, or VABS-II relative to severity asassessed prior to initiating the treatment, and wherein the GI symptomseverity is reduced by at least 40% after a less than 12-week treatmentas assessed by GSRS relative to severity as assessed prior to initiatingthe treatment.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. As used herein, the term “substantially” as in, for example,the phrase “substantially all peptides of an array,” refers to at least90%, preferably at least 95%, more preferably at least 99%, and mostpreferably at least 99.9%, of the peptides of an array. Other uses ofthe term “substantially” involve an analogous definition.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the disclosure.

As used herein, the term “treating” refers to (i) completely orpartially inhibiting a disease, disorder or condition, for example,arresting its development; (ii) completely or partially relieving adisease, disorder or condition, for example, causing regression of thedisease, disorder and/or condition; or (iii) completely or partiallypreventing a disease, disorder or condition from occurring in a patientthat may be predisposed to the disease, disorder and/or condition, buthas not yet been diagnosed as having it. Similarly, “treatment” refersto both therapeutic treatment and prophylactic or preventative measures.In the context of autism spectrum disorder, “treat” and “treating”encompass alleviating, ameliorating, delaying the onset of, inhibitingthe progression of, or reducing the severity of one or more symptomsassociated with an autism spectrum disorder.

As used herein, a “subject” can be a human or animal including, but notlimited to, a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent,e.g., rats and mice, and primate, e.g., monkey. Preferred subjects arehuman subjects. The human subject may be a pediatric, adult or ageriatric subject.

As used herein, a “microbiota” and “flora” refer to a community ofmicrobes that live in or on a subject's body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)). A “fecal microbiota” or“fecal microbiota preparation” refers to a community of microbes presentin or prepared from a subject's feces. A non-selective fecal microbiotarefers to a community or mixture of fecal microbes derived from adonor's fecal sample without selection and substantially resemblingmicrobial constituents and population structure found in such fecalsample.

As used herein, “therapeutically effective amount” or “pharmaceuticallyactive dose” refers to an amount of a composition which is effective intreating the named disease, disorder or condition.

As used herein, “isolated” or “purified” refers to a bacterium or otherentity or substance that has been (1) separated from at least some ofthe components with which it was associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolated orpurified bacteria can be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they were initiallyassociated.

As used herein, the terms “non-pathogenic” in reference to a bacteriumor any other organism or entity includes any such organism or entitythat is not capable of causing or affecting a disease, disorder orcondition of a host organism containing the organism or entity.

As used herein, “spore” or a population of “spores” includes bacteria(or other single-celled organisms) that are generally viable, moreresistant to environmental influences such as heat and bacteriocidalagents than vegetative forms of the same bacteria, and typically capableof germination and out-growth. “Spore-formers” or bacteria “capable offorming spores” are those bacteria containing the genes and othernecessary abilities to produce spores under suitable environmentalconditions.

As used herein, “colony forming units” (cfu) refers to an estimate ofthe number of viable microorganism cells in a given sample. The numberof cfu can be assessed by counting the number of colonies on an agarplate as in standard methods for determining the number of viablebacterial cells in a sample.

As used herein, “viable” means possessing the ability to multiply. Theviability of bacterial populations can be monitored as a function of themembrane integrity of the cell. Cells with a compromised membrane areconsidered to be dead or dying, whereas cells with an intact membraneare considered live. For example, SYTO 9 and propidium iodide are usedto stain and differentiate live and dead bacteria. See Stocks, CytometryA. 2004 October; 61(2):189-95. Cell viability can also be evaluated viamolecular viability analyses, e.g., a PCR-based approach, which candifferentiate nucleic acids associated with viable cells from thoseassociated with inactivated cells. See Cangelosi and Mescheke, ApplEnviron Microbiol. 2014 October; 80(19): 5884-5891.

As used herein, “Shannon Diversity Index” refers to a diversity indexthat accounts for abundance and evenness of species present in a givencommunity using the formula H=−Σ_(i=1) ^(R)p_(i) ln p_(i), where H isShannon Diversity Index, R is the total number of species in thecommunity, and p_(i) is the proportion of R made up of the ith species.Higher values indicate diverse and equally distributed communities, anda value of 0 indicates only one species is present in a given community.For further reference, see Shannon and Weaver, (1949) The mathematicaltheory of communication. The University of Illinois Press, Urbana. 117pp.

As used herein, “antibiotic” refers to a substance that is used to treatand/or prevent bacterial infection by killing bacteria, inhibiting thegrowth of bacteria, or reducing the viability of bacteria.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that ischaracterized by impairments in social interaction and communication,restricted interests, and repetitive behavior. Individuals on the autismspectrum experience widely varying degrees and types of impairments,from mild to severe. Although early detection and interventions areencouraged to maximize the benefits and reduce the severity of thesymptoms, individuals of any age can benefit from interventions andtherapies that can reduce symptoms and increase skills and abilities.Appropriate subjects for the methods described herein include, withoutlimitation, humans diagnosed as having or suspected of having autismspectrum disorder. In some cases, appropriate subjects for the methodsprovided herein are considered to be at increased risk (e.g., moderateor high risk) of developing ASD. In some cases, the subject has beendiagnosed as having a condition meeting diagnostic criteria for ASD asset forth in the DSM-V. In other cases, the subject has awell-established DSM-IV diagnosis of autistic disorder, Asperger'sdisorder, or pervasive developmental disorder not otherwise specified(PDD-NOS).

The methods provided herein result in, or are aimed at achieving adetectable improvement in one or more indicators or symptoms of ASDincluding, without limitation, including, but not limited to, changes ineye tracking, skin conductance and/or EEG measurements in response tovisual stimuli, difficulties engaging in and responding to socialinteraction, verbal and nonverbal communication problems, repetitivebehaviors, intellectual disability, difficulties in motor coordination,attention issues, sleep disturbances, and physical health issues such asgastrointestinal disturbances.

Several screening instruments are known in the art for evaluating asubject's social and communicative development and thus can be used asaids in screening for and detecting changes in the severity ofimpairment in communication skills, social interactions, and restricted,repetitive and stereotyped patterns of behavior characteristic of autismspectrum disorder. Evaluation can include neurologic and geneticassessment, along with in-depth cognitive and language testing.Additional measures developed specifically for diagnosing and assessingautism include the Autism Diagnosis Interview-Revised (ADI-R), theAutism Diagnostic Observation Schedule (ADOS-G) and the Childhood AutismRating Scale (CARS).

According to CARS, evaluators rate the subject on a scale from 1 to 4 ineach of 15 areas: Relating to People; Imitation; Emotional Response;Body Use; Object Use; Adaptation to Change; Visual Response; ListeningResponse; Taste, Smell, and Touch Response and Use; Fear; VerbalCommunication; Nonverbal Communication; Activity; Level and Consistencyof Intellectual Response; and General Impressions.

A second edition of CARS, known as the Childhood Autism Rating Scale—2or CARS-2, was developed by Schopler et al. (Childhood Autism RatingScale—Second edition (CARS2): Manual. Los Angeles: Western PsychologicalServices, 2010). The original CARS was developed primarily withindividuals with co-morbid intellectual functioning and was criticizedfor not accurately identifying higher functioning individuals with ASD.CARS-2 retained the original CARS form for use with younger or lowerfunctioning individuals (now renamed the CARS2-ST for “Standard Form”),but also includes a separate rating scale for use with higherfunctioning individuals (named the CARS2-HF for “High Functioning”) andan unscored information-gathering scale (“Questionnaire for Parents orCaregivers” or CARS2-QPC) that has utility for making CARS2ST andCARS2-HF ratings.

Another symptom rating instrument useful for assessing changes insymptom severity before, during, or following treatment according to amethod provided herein is the Aberrant Behavior Checklist (ABC). SeeAman et al., Psychometric characteristics of the aberrant behaviorchecklist. Am J Ment Defic. 1985 March; 89(5):492-502. The ABC is asymptom rating checklist used to assess and classify problem behaviorsof children and adults in a variety of settings. The ABC includes 58items that resolve onto five subscales: (1) irritability/agitation, (2)lethargy/social withdrawal, (3) stereotypic behavior, (4)hyperactivity/noncompliance, and (5) inappropriate speech.

The present inventors observed that autistic individuals, regardless ofthe presence or absence of comorbid gastrointestinal distress, havefewer species of gut bacteria as compared to neurotypical individuals.The present inventors also found that restoring the species diversity ofgut bacteria helps to treat autistic symptoms in patients in needthereof. In one aspect, this application provides a method for treatingan autism spectrum disorder (ASD) in a subject in need thereof, themethod comprising administering to the subject an amount of apharmaceutical composition effective for treating the ASD, where thepharmaceutical composition comprises a fecal microbe preparation, wherethe subject exhibits at least a 10% reduction in ASD symptom severityafter the treatment as compared to before initiating the treatment. Inone aspect, ASD symptom severity is assessed by Childhood Autism RatingScale (CARS). In another aspect, ASD symptom severity is assessed byChildhood Autism Rating Scale 2-Standard Form (CARS2-ST). In a furtheraspect, ASD symptom severity is assessed by Childhood Autism RatingScale 2-High Functioning (CARS2-HF). In one aspect, ASD symptom severityis assessed by Aberrant Behavior Checklist (ABC). In another aspect, ASDsymptom severity is assessed by Social Responsiveness Scale (SRS). Inanother aspect, ASD symptom severity is assessed by Vineland AdaptiveBehavior Scale II (VABS-II). In one aspect, a treatment results in animprovement of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%based on the Leiter International Performance Scale (see Roid, G. H., &Miller, L. J. (1997). Leiter International Performance Scale—Revised.Wood Dale, Ill.: Stoelting) in an ASD patient. In another aspect, aLeiter score improvement is measured after at least 8, 16, 24, 32, 40,50, 60, or 80 weeks of treatment and compared to a Leiter score prior tothe treatment.

One of ordinary skill in the art understands that the foregoingassessment systems are only exemplary tools for evaluating ASD-relatedsocial and cognitive symptoms. Other similar tools can be used ordesigned to evaluate core ASD-related symptoms. For example, in oneaspect, a treatment results in an improvement of at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% based on Autism Treatment EvaluationChecklist (ATEC). See Rimland and Edelson: Autism Treatment EvaluationChecklist: Statistical Analyses. Autism Research Institute 2000. Inanother aspect, a treatment results in an improvement of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% based on PervasiveDevelopmental Disorders Behavior Inventory (PDD-BI). See Cohen et al.,The PDD Behavior Inventory: a rating scale for assessing response tointervention in children with pervasive developmental disorder. J AutismDev Disord. 2003 33(1):31-45. In yet another aspect, a treatment resultsin an improvement of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% based on Severity of Autism Scale (SAS). See Adams et al., Theseverity of autism is associated with toxic metal body burden and redblood cell glutathione levels. J Toxicol. 2009, 2009:532640. In afurther aspect, an improvement of autism-related symptoms or an symptomseverity reduction is assessed based on any one of the system or scalementioned in Aman et al., Outcome Measures for Clinical Drug Trials inAutism, CNS Spectr. 9(1): 36-47 (2004). In a further aspect, animprovement of autism-related symptoms or an symptom severity reductionis assessed based on any one of the symptom characterization systemslisted in Table 1. In one aspect, an symptom improvement over any one ofthe foregoing systems is measured after at least 8, 16, 24, 32, 40, 50,60, or 80 weeks of treatment and compared to a Leiter score prior to thetreatment. In one aspect, an symptom improvement over any one of theforegoing systems is measured after discontinuing treatment for at least2, 4, 6, 8, 10 or more weeks and compared to a measurement prior to thetreatment.

TABLE 1 Selected outcome measures that can be used to monitor coreASD-related social and cognitive symptoms. Validated Outcome MeasuresDescription Tool Autism Symptoms Rater ADOS The Autism DiagnosticObservation Trained Schedule (ADOS) is a gold standards Examinerinstrument for diagnosing ASD with the largest evidence base and highestsensitivity and specificity OACIS The Ohio Autism Clinical ImpressionScale Clinician was developed to be sensitive to subtle, butclinically-meaningful changes in core and associated ASD symptoms usinga focused scaling system that assesses severity and improvement in ASDbehaviors similar to the widely used Clinical Global Impression Scale.SRS The Social Responsiveness Scale is a Parent or standardized andvalidated quantitative Teacher scale that measures the severity and typeof social impairments that are characteristic of ASD SCQ SocialCommunication Questionnaire Parent or is brief instrument that evaluatesTeacher communication skills and social functioning. Both the currentand lifetime editions will be used as appropriate AIM The Autism ImpactMeasure is a recently Parent developed parent-report measure thatassesses both frequency and impact of current core ASD symptoms duringthe past 2-weeks. Initial studies have demonstrated excellentpsychometric properties and construct validity Behavior ABC The AberrantBehavior Checklist is a validated Parent or questionnaire that ratessymptoms Teacher of hyperactivity, irritability, lethargy, andstereotypic behavior in individuals with developmental disabilities. Ithas been used in multiple clinical trials in ASD and has convergent anddivergent validity CBCL Child Behavior Checklist is an easy to Parent orcomplete standardized questionnaire that Teacher assesses a wide rangeof behaviors associated with ASD symptoms, including anxiety,depression, withdraw, sleep problems, somatic problems, and aggressiveand destructive behavior BASC The Behavioral Assessment System forParent or Children provides scales of cognition Teacher function,behavior, social function, and academic problems. This scale measures awide range of behaviors including hyperactivity, attention, depression,anxiety, and executive function. Language CELF The Clinical Evaluationsof Language Trained Fundamentals is one of the only Examinerstandardized, well-validated language assessment instruments that spansthe age range of most participants (using both CELF-preschool-2 andCELF-4). It assesses a wide range of language skills that are onlypartially measured by other language tests, including high-levellanguage skills that are abnormal in individuals with ASD, such aslanguage pragmatics and has been used in several recent studies focusingon core language deficits in ASD PLS The Preschool Language Scale-4 isused in Trained conjunction with the CELF since it is Examiner also astandardized, well-validated language assessment instrument and canmeasure subtle changes in language in children with poor languageabilities Adaptive Behavior VABS The Vineland Adaptive Behavior Scale isTrained a widely used standardized, well- Interviewer validatedassessment tool for children with developmental delays that measuresfunctional abilities within several domains. It is particularly usefulfor children with intellectual disability which commonly co-occurs withASD and has valid measures of social impairments in children with ASDIntellect Leiter-R The Leiter-R, due to its non-verbal nature, Trainedis an excellent unbiased measure of Examiner intellect when languageimpairment exists. It assesses a wide range of ages (2-21 years) andcontains attention and memory batteries which are skills often disruptedin ASD. The Leiter-R is designed to measure growth in all domains itassesses, making it sensitive to change due to treatment. Studies haveshown good psychometric properties and verified that it is generallyrecommended for use in children with ASD WISC/ The Wechsler IntelligenceScale for Children Trained WPPSI is one of the oldest and most widelyExaminer used tests of intelligence for children. For children youngerthan 6 years the Wechsler Preschool and Primary Scale of Intelligencetest is used. One disadvantage when using this with children with ASD isits reliance on language.

In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% reduction in ASD symptom severity after 2 or moreweeks of treatment as compared to before initiating the treatment, wherethe ASD symptom severity is assessed by a method selected from the groupconsisting of CARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. In oneaspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% reduction in ASD symptom severity after 4 or more weeks oftreatment as compared to before initiating the treatment, where the ASDsymptom severity is assessed by a method selected from the groupconsisting of CARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. In oneaspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% reduction in ASD symptom severity after 6 or more weeks oftreatment as compared to before initiating the treatment, where the ASDsymptom severity is assessed by a method selected from the groupconsisting of CARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. In oneaspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% reduction in ASD symptom severity after 8 or more weeks oftreatment as compared to before initiating the treatment, where the ASDsymptom severity is assessed by a method selected from the groupconsisting of CARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II.

In another aspect, a treatment achieves between 10% and 20%, between 10%and 30%, between 10% and 40%, between 10% and 50%, between 10% and 60%,between 10% and 70%, between 10% and 80%, between 10% and 90%, between20% and 30%, between 20% and 40%, between 20% and 50%, between 20% and60%, between 20% and 70%, between 20% and 80%, between 20% and 90%,between 30% and 40%, between 30% and 50%, between 30% and 60%, between30% and 70%, between 30% and 80%, between 30% and 90%, between 40% and50%, between 40% and 60%, between 40% and 70%, between 40% and 80%,between 40% and 90%, between 50% and 60%, between 50% and 70%, between50% and 80%, or between 50% and 90% reduction in ASD symptom severityafter 8 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In another aspect, a treatment achieves between 10%and 90%, between 20% and 80%, between 30% and 70%, or between 40% and60% reduction in ASD symptom severity after 8 or more weeks of treatmentas compared to before initiating the treatment, where the ASD symptomseverity is assessed by a method selected from the group consisting ofCARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. In another aspect, atreatment achieves between 10% and 90%, between 20% and 80%, between 30%and 70%, or between 40% and 60% reduction in ASD symptom severity after12 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In another aspect, a treatment achieves between 10%and 90%, between 20% and 80%, between 30% and 70%, or between 40% and60% reduction in ASD symptom severity after 18 or more weeks oftreatment as compared to before initiating the treatment, where the ASDsymptom severity is assessed by a method selected from the groupconsisting of CARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. Inanother aspect, a treatment achieves between 10% and 90%, between 20%and 80%, between 30% and 70%, or between 40% and 60% reduction in ASDsymptom severity after 24 or more weeks of treatment as compared tobefore initiating the treatment, where the ASD symptom severity isassessed by a method selected from the group consisting of CARS,CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II.

In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 8,12, 16, 20, 24, or 28 weeks after discontinuing the treatment, where theASD symptom severity is assessed by CARS. In one aspect, a treatmentachieves at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%reduction in ASD symptom severity and substantially maintains thesymptom severity reduction for at least 8, 12, 16, 20, 24, or 28 weeksafter discontinuing the treatment, where the ASD symptom severity isassessed by CARS2-ST. In one aspect, a treatment achieves at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptomseverity and substantially maintains the symptom severity reduction forat least 8, 12, 16, 20, 24, or 28 weeks after discontinuing thetreatment, where the ASD symptom severity is assessed by CARS2-HF. Inone aspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% reduction in ASD symptom severity and substantiallymaintains the symptom severity reduction for at least 8, 12, 16, 20, 24,or 28 weeks after discontinuing the treatment, where the ASD symptomseverity is assessed by ABC. In one aspect, a treatment achieves atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASDsymptom severity and substantially maintains the symptom severityreduction for at least 8, 12, 16, 20, 24, or 28 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby SRS. In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 8,12, 16, 20, 24, or 28 weeks after discontinuing the treatment, where theASD symptom severity is assessed by VABS-II.

In one aspect, an ASD subject being treated exhibits no gastrointestinal(GI) symptom prior to initiating a treatment. In another aspect, an ASDsubject being treated exhibits one or more GI symptoms prior toinitiating a treatment. In one aspect, an ASD subject being treatedexhibits at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reductionin GI symptom severity after a treatment as compared to beforeinitiating the treatment. In one aspect, GI symptom severity is assessedby the Gastrointestinal Symptom Rating Scale (GSRS). In another aspect,a treatment achieves between 20% and 30%, between 20% and 40%, between20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and80%, between 20% and 90%, between 30% and 40%, between 30% and 50%,between 30% and 60%, between 30% and 70%, between 30% and 80%, between30% and 90%, between 40% and 50%, between 40% and 60%, between 40% and70%, between 40% and 80%, between 40% and 90%, between 50% and 60%,between 50% and 70%, between 50% and 80%, or between 50% and 90%reduction in GI symptom severity in an ASD patient after 8 or more weeksof treatment as compared to before initiating the treatment, where theGI symptom severity is assessed by GSRS.

In one aspect, a symptom severity reduction (e.g., for ASD symptoms, GIsymptoms, or both) is ongoing during a treatment or sustained afterfinishing or discontinuing a treatment. In one aspect, a symptomseverity reduction (e.g., for ASD symptoms, GI symptoms, or both) isassessed at a specific time point during or post treatment, e.g., about2, 4, 6, 8, 12, 18, 24, 32, 40, 48 weeks after initiating a treatment,or about 2, 4, 6, 8, 12, 18, 24, 32, 40, 48 weeks after finishing ordiscontinuing a treatment.

In one aspect, a method further comprises administering an antibiotic toa subject prior to administering a pharmaceutical composition comprisinga fecal microbe preparation. In another aspect, a method furthercomprises subjecting a subject to a bowel cleanse.

In another aspect, a pharmaceutical composition used herein comprises anon-selective and substantially complete fecal microbiota supplementedwith one or more viable, non-pathogenic microorganisms selected from thegroup consisting of Prevotella, Desulfovibrio, Copprococcus, andClostridium. In another aspect, a pharmaceutical composition used hereincomprises a synthetic fecal composition of predetermined flora. Inanother aspect, a pharmaceutical composition used herein comprises apredetermined flora comprises a preparation of viable flora inproportional content that resembles a normal healthy human fecal floraand comprises no antibiotic resistant populations. In another aspect, apharmaceutical composition used herein is administered as a solid dosageform selected from the group consisting of capsule, tablet, powder, andgranule. In another aspect, a pharmaceutical composition used herein isformulated as an acid resistant capsule.

In another aspect, provided herein is a method of treating an autismspectrum disorder in a human subject. In exemplary aspects, the methodcomprises or consists essentially of the following steps: administeringan antibiotic to a human subject; subjecting the human subject to abowel cleanse; and administering purified fecal microbiota to the humansubject, wherein an autism spectrum disorder is treated in the humansubject.

In exemplary aspects, treating ASD comprises alleviating, ameliorating,delaying the onset of, inhibiting the progression of, or reducing theseverity of one or more, two or more, three or more, four or more, fiveor more, six or more, seven or more, eight or more symptomscharacteristic of ASD. In one aspect, a treatment alleviates,ameliorates, delays the onset of, inhibites the progression of, orreduces the severity of one or more social and cognitive coreASD-related symptoms. In some aspects, the symptom(s) is selected fromthe group consisting of: (i) insistence on sameness or resistance tochange; (ii) difficulty in expressing needs; (iii) repeating words orphrases in place of normal, responsive language; (iv) laughing, crying,showing distress for reasons not apparent to others; (v) prefers to bealone or aloof manner; (vi) tantrums; (vii) difficulty in mixing withothers; (viii) may not want to cuddle or be cuddled; (ix) little or noeye contact; (x) unresponsive to normal teaching methods; (xi) sustainedodd play; (xii) apparent over-sensitivity or under-sensitivity to pain;(xiii) little or no real fears of danger; (xiv) noticeable physicalover-activity or extreme under-activity; (xv) uneven gross/fine motorskills; and/or (xvi) non-responsiveness to verbal cues. In some aspects,the symptom(s) is selected from the group consisting of compulsivebehavior, ritualistic behavior, restricted behavior, stereotypy,sameness, or self-injury. The methods described here can lead toimprovement of any combination of the foregoing symptoms.

In exemplary aspects, the human subject exhibits a significant reductionin autism symptom severity as assessed according to a ASD rating scale.In some cases, for example, the human subject exhibits at least a 10% or20% reduction in autism symptom severity as assessed by the ChildhoodAutism Rating Scale (CARS) relative to severity as assessed prior toinitiating the method.

Subjects appropriate for treatment according to a method provided hereinmay not present with or report gastrointestinal distress symptoms priorto initiating a method as provided herein. In some cases, for example, ahuman subject appropriate for treatment according to a method providedherein manifests no gastrointestinal symptoms prior to or at the time atwhich treatment is begun. In one aspect, an ASD subject treated hereinexhibit one or more or two or more GI symptoms selected from the groupconsisting of abdominal pain, reflux, indigestion, irritable bowelsyndrome, chronic persistent diarrhoea, diarrhoea, flatulence,constipation, and alternating constipation/diarrhoea.

Regardless of the presence or absence of gastrointestinal distresssymptoms, human subjects appropriate for the methods provided hereintypically have significantly fewer species of gut bacteria before saidmethod of treatment as compared to a neurotypical human. In some cases,the human subject to be treated by the method exhibits at least about20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% fewer species of gut bacterialprior to administration of the purified fecal microbiota dosage ascompared to a neurotypical human.

Also provided herein are methods for reducing autism severity in anautistic human subject. In exemplary aspects, the method comprises orconsists essentially of the following steps: orally-administering anon-absorbable antibiotic to an autistic human subject; subjecting theautistic human subject to a bowel cleanse; and administering purifiedfecal microbiota from a neurotypical human donor to the human subject,wherein the human subject exhibits a significant reduction in autismsymptom severity as assessed by the Childhood Autism Rating Scale (CARS)after said method as compared to before initiating the method. In somecases, the human subject exhibits at least a 10% or 20% reduction inautism symptom severity as assessed by the Childhood Autism Rating Scale(CARS) relative to severity as assessed prior to initiating the method.

In one aspect, a fecal microbiota preparation used in a method describedhere comprises a donor's entire or substantially complete microbiota. Inone aspect, a fecal microbiota preparation comprises a non-selectivefecal microbiota. In another aspect, a fecal microbiota preparationcomprises an isolated or purified population of live non-pathogenicfecal bacteria. In a further aspect, a fecal microbiota preparationcomprises a non-selective and substantially complete fecal microbiotapreparation from a single donor. In another aspect, a therapeuticcomposition used herein comprises a mixture of live, non-pathogenic,synthetic bacteria or live, non-pathogenic, purified or extracted, fecalmicrobiota.

In one aspect, the preparation of a fecal microbiota preparationinvolves a treatment selected from the group consisting of ethanoltreatment, detergent treatment, heat treatment, irradiation, andsonication, or a combination thereof. In one aspect, the preparation ofa fecal microbiota preparation involves no treatment selected from thegroup consisting of ethanol treatment, detergent treatment, heattreatment, irradiation, and sonication. In one aspect, the preparationof a fecal microbiota preparation involves a separation step selectedfrom the group consisting of filtering, sieving, density gradients,filtration, chromatography, and a combination thereof. In one aspect,the preparation of a fecal microbiota preparation does not require oneor more separation steps selected from the group consisting offiltering, sieving, density gradients, filtration, and chromatography.In one aspect, a fecal microbiota preparation is substantially free ofnon-living matter. In one aspect, a fecal microbiota preparation issubstantially free of acellular material selected from the groupconsisting of residual fiber, DNA, viral coat material, and non-viablematerial. In one aspect, a fecal microbiota preparation is substantiallyfree of eukaryotic cells from the fecal microbiota's donor.

In one aspect, the present disclosure provides a method for treating ASDin a subject in need thereof, where the method comprises administeringto the subject a pharmaceutically active dose of a therapeuticcomposition described herein. In one aspect, the present disclosureprovides a method for treating ASD in a subject in need thereof, wherethe method comprises administering daily to the subject apharmaceutically active dose of a therapeutic composition describedherein. In one aspect, a therapeutic composition is administered to apatient in need thereof at least once daily for at least two consecutivedays. In one aspect, a therapeutic composition is administered at leastonce daily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15consecutive days. In another aspect, a therapeutic composition isadministered at least once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 consecutive weeks. In one aspect, a therapeuticcomposition is administered at least once daily for at most 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days orweeks. In another aspect, a therapeutic composition is administered atleast once daily for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12consecutive weeks or months. In a further aspect, a therapeuticcomposition is administered at least once for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 consecutive months or years, chronically for asubject's entire life span, or an indefinite period of time.

In one aspect, a therapeutic composition is administered to a patient inneed thereof at least twice daily for at least two consecutive days. Inone aspect, a therapeutic composition is administered at least twicedaily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15consecutive days. In another aspect, a therapeutic composition isadministered at least twice daily for at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 consecutive weeks. In one aspect, a therapeuticcomposition is administered at least twice daily for at most 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days orweek. In another aspect, a therapeutic composition is administered atleast twice daily for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12consecutive weeks or months. In a further aspect, a therapeuticcomposition is administered at least twice for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 consecutive months or years, chronically for asubject's entire life span, or an indefinite period of time.

In one aspect, a therapeutic composition is administered to a patient inneed thereof at least three times daily for at least two consecutivedays. In one aspect, a therapeutic composition is administered at leastthree times daily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15 consecutive days. In another aspect, a therapeutic composition isadministered at least three times daily for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 consecutive weeks. In one aspect, a therapeuticcomposition is administered at least three times daily for at most 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutivedays or weeks. In another aspect, a therapeutic composition isadministered at least three times daily for at most 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 consecutive weeks or months. In a further aspect, atherapeutic composition is administered at least three times for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive months oryears, chronically for a subject's entire life span, or an indefiniteperiod of time.

In one aspect, the present disclosure provides a method for treating ASDin a subject in need thereof, where the method comprises administeringorally to the subject a pharmaceutically active dose of a therapeuticcomposition comprising live, non-pathogenic, synthetic bacterial mixtureor live, non-pathogenic, purified or extracted, fecal microbiota, wherethe dose is administered at a dosing schedule of at least once or twicedaily for at least three consecutive days or weeks. In another aspect, adose is administered at least once, twice, or three times daily for aperiod between 1 and 12 weeks, between 2 and 12 weeks, between 3 and 12weeks, between 4 and 12 weeks, between 5 and 12 weeks, between 6 and 12weeks, between 7 and 12 weeks, between 8 and 12 weeks, between 9 and 12weeks, between 10 and 12 weeks, between 1 and 2 weeks, between 2 and 3weeks, between 3 and 4 weeks, between 4 and 5 weeks, between 5 and 6weeks, between 6 and 7 weeks, between 7 and 8 weeks, between 8 and 9weeks, between 9 and 10 weeks, or between 10 and 11 weeks.

In one aspect, the present disclosure provides a method for treating ASDin a subject in need thereof by administering a pharmaceuticalcomposition described herein, where the method comprises a first dosingschedule followed by a second dosing schedule. In one aspect, a firstdosing schedule comprises a treatment or induction dose. In one aspect,a first dosing schedule comprises a continuous dosing schedule. Inanother aspect, a second dosing schedule comprises a maintenance doselower than or equal to a pharmaceutically active dose of a first dosingschedule. In another aspect, a second dosing schedule lasts for at leastabout 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72, or 96 months. In oneaspect, a second dosing schedule lasts permanently, for a treatedsubject's entire life span, or an indefinite period of time. In oneaspect, a second dosing schedule is a continuous dosing schedule. Inanother aspect, a second dosing schedule is an intermittent dosingschedule. In a further aspect, a second dosing schedule is anintermittent dosing schedule comprising a treatment period of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days followed by aresting period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days. In another aspect, a second dosing schedule comprisesadministering a second dose (e.g., a maintenance dose) every other day,every two days, or every 3, 4, 5, 6, 7, 8 days. In another aspect, amaintenance dose is administered for an extended period of time with orwithout titration (or otherwise changing the dosage or dosing schedule).In one aspect, the interval between a first and a second dosing scheduleis at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. Inanother aspect, a second dosing schedule (e.g., a maintenance dose)comprises a dosage about 2, 5, 10, 50, 100, 200, 400, 800, 1000, 5000 ormore folds lower than the dosage used in a first dosing schedule (e.g.,an initial treatment dose). In another aspect, a second dosing schedule(e.g., a maintenance dosing schedule) has an equal or lower dosingfrequency than a first dosing schedule (e.g., an initial treatmentdosing schedule). In another aspect, a second dosing schedule (e.g., amaintenance dosing schedule) has a higher dosing interval than a firstdosing schedule (e.g., an initial treatment dosing schedule).

In one aspect, a first or second dosing schedule used in a method can beonce-a-week, twice-a-week, or thrice-a-week. The term “once-a-week”means that a dose is administered once in a week, preferably on the sameday of each week. “Twice-a-week” means that a dose is administered twotimes in a week, preferably on the same two days of each weekly period.“Thrice-a-week” means that a dose is administered three times in a week,preferably on the same three days of each weekly period.

In one aspect, a subject being treated is a subject already with adisorder (e.g., ASD). Administration of a disclosed therapeuticcomposition to clinically, asymptomatic human subject who is geneticallypredisposed or prone to a disorder (e.g., ASD) is also useful inpreventing the onset of clinical symptoms. A human subject geneticallypredisposed or prone to ASD can be a human subject having a close familymember or relative exhibiting or having suffered a disorder (e.g., ASD).In another aspect, a subject being treated is a subject in which ASD isto be prevented. In another aspect, a subject being treated ispredisposed or susceptible to a disorder (e.g., ASD). In another aspect,a subject being treated is a subject diagnosed as having a disorder(e.g., ASD). In one aspect, a subject being treated is a patient in needthereof.

In one aspect, a subject being treated is a human patient. In oneaspect, a patient is a male patient. In one aspect, a patient is afemale patient. In one aspect, a patient is a premature newborn. In oneaspect, a patient is a term newborn. In one aspect, a patient is aneonate. In one aspect, a patient is an infant. In one aspect, a patientis a toddler. In one aspect, a patient is a young child. In one aspect,a patient is a child. In one aspect, a patient is an adolescent. In oneaspect, a patient is a pediatric patient. In one aspect, a patient is ageriatric patient. In one aspect, a human patient is a child patientbelow about 18, 15, 12, 10, 8, 6, 4, 3, 2, or 1 year old. In anotheraspect, a human patient is an adult patient. In another aspect, a humanpatient is an elderly patient. In a further aspect, a human patient is apatient above about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,or 95 years old. In another aspect, a patient is about between 1 and 5,between 2 and 10, between 3 and 18, between 21 and 50, between 21 and40, between 21 and 30, between 50 and 90, between 60 and 90, between 70and 90, between 60 and 80, or between 65 and 75 years old. In oneaspect, a patient is a young old patient (65-74 years). In one aspect, apatient is a middle old patient (75-84 years). In one aspect, a patientis an old patient (>85 years).

In one aspect, a method comprises administering a therapeuticcomposition orally, by enema, or via rectal suppository. In one aspect,a pharmaceutical composition is formulated as a geltab, pill,microcapsule, capsule, or tablet. In one aspect, a therapeuticcomposition is formulated as an enteric coated capsule or microcapsule,acid-resistant capsule or microcapsule, or formulated as part of oradministered together with a food, a food additive, a dairy-basedproduct, a soy-based product or a derivative thereof, a jelly, or ayogurt. In another aspect, a therapeutic composition is formulated as anacid-resistant enteric coated capsule. A therapeutic composition can beprovided as a powder for sale in combination with a food or drink. Afood or drink can be a dairy-based product or a soy-based product. Inanother aspect, a food or food supplement contains enteric-coated and/oracid-resistant microcapsules containing a therapeutic composition.

In an aspect, a therapeutic composition comprises a liquid culture. Inanother aspect, a therapeutic composition is lyophilized, pulverized andpowdered. It may then be infused, dissolved such as in saline, as anenema. Alternatively the powder may be encapsulated as enteric-coatedand/or acid-resistant capsules for oral administration. These capsulesmay take the form of enteric-coated and/or acid-resistant microcapsules.A powder can preferably be provided in a palatable form forreconstitution for drinking or for reconstitution as a food additive. Ina further aspect, a food is yogurt. In one aspect, a powder may bereconstituted to be infused via naso-duodenal infusion.

In another aspect, a therapeutic composition is in a liquid, frozen,freeze-dried, spray-dried, lyophilized, or powder formulation. In afurther aspect, a therapeutic composition is formulated as a delayed orgradual enteric release form. In another aspect, a therapeuticcomposition comprises an excipient, a saline, a buffer, a bufferingagent, or a fluid-glucose-cellobiose agar (RGCA) media.

In one aspect, a therapeutic composition further comprises an acidsuppressant, an antacid, an H2 antagonist, a proton pump inhibitor or acombination thereof. In one aspect, a therapeutic compositionsubstantially free of non-living matter. In another aspect, atherapeutic composition substantially free of acellular materialselected from the group consisting of residual fiber, DNA, viral coatmaterial, and non-viable material.

In one aspect, a therapeutic composition comprises a cryoprotectant. Inanother aspect, a cryoprotectant comprises, consisting essentially or,or consisting of polyethylene glycol, skim milk, erythritol, arabitol,sorbitol, glucose, fructose, alanine, glycine, proline, sucrose,lactose, ribose, trehalose, dimethyl sulfoxide (DMSO), glycerol, or acombination thereof.

In another aspect, a therapeutic composition comprises a lyoprotectant.In one aspect, the same substance or the same substance combination isused as both a cryoprotectant and a lyoprotectant. Exemplarylyoprotectants include sugars such as sucrose or trehalose; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher sugar alcohols, e.g. glycerin, erythritol, glycerol,arabitol, xylitol, sorbitol, and mannitol; propylene glycol;polyethylene glycol; Pluronics; and combinations thereof. In one aspect,a lyoprotectant is a non-reducing sugar, such as trehalose or sucrose.In one aspect, a cryoprotectant or a lyoprotectant consistingessentially of, or consisting of, one or more substances mentioned inthis paragraph and the paragraph above.

In one aspect, a lyophilized formulation comprises trehalose. In oneaspect, a lyophilized formulation comprises 2% to 30%, 3% to 25%, 4% to20%, 5% to 15%, 6% to 10%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 15%,or 2% to 10% trehalose. In one aspect, a lyophilized formulationcomprises at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15%trehalose. In one aspect, a lyophilized formulation comprises at most2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% trehalose. In one aspect, alyophilized formulation comprises about 5% trehalose. In one aspect, alyophilized formulation comprises trehalose and sucrose. In one aspect,a lyophilized formulation comprises between about 8% to 12% trehalosewith between about 1.5% to 3.5% sucrose and between about 0.5% to 1.5%NaCl.

In one aspect, a therapeutic composition also comprises or issupplemented with a prebiotic nutrient selected from the groupconsisting of polyols, fructooligosaccharides (FOSs), oligofructoses,inulins, galactooligosaccharides (GOSs), xylooligosaccharides (XOSs),polydextroses, monosaccharides, tagatose, and/or mannooligosaccharides.

In one aspect, a method further comprises pretreating a subject with anantibiotic composition prior to administering a therapeutic bacterial ormicrobiota composition. In one aspect, an antibiotic compositioncomprises an antibiotic selected from the group consisting of rifabutin,clarithromycin, clofazimine, vancomycin, rifampicin, nitroimidazole,chloramphenicol, and a combination thereof. In another aspect, anantibiotic composition comprises an antibiotic selected from the groupconsisting of rifaximin, rifamycin derivative, rifampicin, rifabutin,rifapentine, rifalazil, bicozamycin, aminoglycoside, gentamycin,neomycin, streptomycin, paromomycin, verdamicin, mutamicin, sisomicin,netilmicin, retymicin, kanamycin, aztreonam, aztreonam macrolide,clarithromycin, dirithromycin, roxithromycin, telithromycin,azithromycin, bismuth subsalicylate, vancomycin, streptomycin,fidaxomicin, amikacin, arbekacin, neomycin, netilmicin, paromomycin,rhodostreptomycin, tobramycin, apramycin, and a combination thereof. Ina further aspect, a method further comprises pretreating a subject withan anti-inflammatory drug prior to administration of a therapeuticbacterial or microbiota composition.

In one aspect, every about 200 mg of a pharmaceutical compositioncomprises a pharmacologically active dose. In one aspect, every about75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000,1500, or 2000 mg of a pharmaceutical composition comprises apharmacologically active dose.

In one aspect, a pharmaceutically active or therapeutic effective dosecomprises at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cfu. In another aspect, a pharmaceutically active therapeuticeffective dose comprises at most about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², or 10¹³ cfu. In a further aspect, a pharmacologically activetherapeutic effective dose is selected from the group consisting of from10⁸ cfu to 10¹⁴ cfu, from 10⁹ cfu to 10¹³ cfu, from 10¹⁰ cfu to 10¹²cfu, from 10⁹ cfu to 10¹⁴ cfu, from 10⁹ cfu to 10¹² cfu, from 10⁹ cfu to10¹¹ cfu, from 10⁹ cfu to 10¹⁰ cfu, from 10¹⁰ cfu to 10¹⁴ cfu, from 10¹⁰cfu to 10¹³ cfu, from 10¹¹ cfu to 10¹⁴ cfu, from 10¹¹ cfu to 10¹³ cfu,from 10¹² cfu to 10¹⁴ cfu, and from 10¹³ cfu to 10¹⁴ cfu. In one aspect,a pharmaceutical composition comprises the foregoing pharmaceuticallyactive or therapeutic effective dose in a unit weight of about 0.2, 0.4,0.6, 0.8 or 1.0 gram, or a unit volume of about 0.2, 0.4, 0.6, 0.8 or1.0 milliliter.

In one aspect, a pharmaceutically active or therapeutic effective dosecomprises at least about 10⁵, 10⁶, 10⁷, 10 ⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cells or spores. In another aspect, a pharmaceutically active ortherapeutic effective dose comprises at most about 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³ total cells or spores. In a furtheraspect, a pharmacologically active or therapeutic effective dose isselected from the group consisting of from 10⁸ to 10¹⁴, from 10⁹ to10¹³, from 10¹⁰ to 10¹², from 10⁹ to 10¹⁴, from 10⁹ to 10¹², from 10⁹ to10¹¹, from 10⁹ to 10¹⁰ from 10¹⁰ to 10¹⁴, from 10¹⁰ to 10¹³, from 10¹¹to 10¹⁴, from 10¹¹ to 10¹³, from 10¹² to 10¹⁴, and from 10¹³ to 10¹⁴cells or spores. In an aspect, the pharmaceutically active ortherapeutic effective dose cell count is directed to live cells. In oneaspect, a pharmaceutical composition comprises the foregoingpharmaceutically active or therapeutic effective dose in a unit weightof about 0.2, 0.4, 0.6, 0.8 or 1.0 gram, or a unit volume of about 0.2,0.4, 0.6, 0.8 or 1.0 milliliter.

In one aspect, a therapeutic composition described and used herecomprises one or more, two or more, three or more, four or more, or fiveor more isolated, purified, or cultured microorganisms selected from thegroup consisting of Clostridium, Bacillus, Collinsella, Bacteroides,Eubacterium, Fusobacterium, Propionibacterium, Lactobacillus,Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas,Peptostreptococcus, Bifidobacterium, Coprococcus, Dorea, and Monilia.

In one aspect, a fecal microbiota preparation described herein comprisesa purified or reconstituted fecal bacterial mixture. In one aspect, afecal microbiota preparation described and used here comprises one ormore, one or more, two or more, three or more, four or more, or five ormore live fecal microorganisms are selected from the group consisting ofAcidaminococcus, Akkermansia, Alistipes, Anaerotruncus, Bacteroides,Bifidobacterium, Blautia, Butyrivibrio, Clostridium, Collinsella,Coprococcus, Corynebacterium, Dorea, Enterococcus, Escherichia,Eubacterium, Faecalibacterium, Haemophilus, Holdemania, Lactobacillus,Moraxella, Parabacteroides, Prevotella, Propionibacterium, Raoultella,Roseburia, Ruminococcus, Staphylococcus, Streptococcus, Subdoligranulum,and Veillonella. In one aspect, a fecal microbiota preparation comprisesone or more, one or more, two or more, three or more, four or more, orfive or more live fecal microorganisms are selected from the groupconsisting of Bacteroides fragilis ssp. vulgatus, Collinsellaaerofaciens, Bacteroides fragilis ssp. thetaiotaomicron,Peptostreptococcus productus II, Parabacteroides distasonis,Faecalibacterium prausnitzii, Coprococcus eutactus, Peptostreptococcusproductus I, Ruminococcus bromii, Bifidobacterium adolescentis, Gemmigerformicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcustorques, Eubacterium rectale, Eubacterium eligens, Bacteroideseggerthii, Clostridium leptum, Bacteroides fragilis ssp. A, Eubacteriumbiforme, Bifidobacterium infantis, Eubacterium rectale, Coprococcuscomes, Pseudoflavonifractor capillosus, Ruminococcus albus, Doreaformicigenerans, Eubacterium hallii, Eubacterium ventriosum I,Fusobacterium russi, Ruminococcus obeum, Eubacterium rectale,Clostridium ramosum, Lactobacillus leichmannii, Ruminococcus callidus,Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacteriumventriosum, Bacteroides fragilis ssp. fragilis, Coprococcus catus,Aerostipes hadrus, Eubacterium cylindroides, Eubacterium ruminantium,Staphylococcus epidermidis, Eubacterium limosum, Tissirella praeacuta,Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridiuminnocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcusflavefaciens, Bacteroides fragilis ssp. ovatus, Fusobacterium nucleatum,Fusobacterium mortiferum, Escherichia coli, Gemella morbillorum,Finegoldia magnus, Streptococcus intermedius, Ruminococcus lactaris,Eubacterium tenue, Eubacterium ramulus, Bacteroides clostridiiformisssp. clostridliformis, Bacteroides coagulans, Prevotella oralis,Prevotella ruminicola, Odoribacter splanchnicus, and Desuifomonas pigra.

In one aspect, a fecal microbiota preparation described and used herelacks or is substantially devoid of one or more, one or more, two ormore, three or more, four or more, or five or more live fecalmicroorganisms are selected from the group consisting ofAcidaminococcus, Akkermansia, Alistipes, Anaerotruncus, Bacteroides,Bifidobacterium, Blautia, Butyrivibrio, Clostridium, Collinsella,Coprococcus, Corynebacterium, Dorea, Enterococcus, Escherichia,Eubacterium, Faecalibacterium, Haemophilus, Holdemania, Lactobacillus,Moraxella, Parabacteroides, Prevotella, Propionibacterium, Raoultella,Roseburia, Ruminococcus, Staphylococcus, Streptococcus, Subdoligranulum,and Veillonella. In one aspect, a fecal microbiota preparation lacks oris substantially devoid of one or more, one or more, two or more, threeor more, four or more, or five or live more fecal microorganisms areselected from the group consisting of Bacteroides fragilis ssp.vulgatus, Collinsella aerofaciens, Bacteroides fragilis ssp.thetaiotaomicron, Peptostreptococcus productus II, Parabacteroidesdistasonis, Faecalibacterium prausnitzii, Coprococcus eutactus,Peptostreptococcus productus I, Ruminococcus bromii, Bifidobacteriumadolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacteriumsiraeum, Ruminococcus torques, Eubacterium rectale, Eubacterium eligens,Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilis ssp. A,Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale,Coprococcus comes, Pseudoflavonifractor capillosus, Ruminococcus albus,Dorea formicigenerans, Eubacterium hallii, Eubacterium ventriosum I,Fusobacterium russi, Ruminococcus obeum, Eubacterium rectale,Clostridium ramosum, Lactobacillus leichmannii, Ruminococcus callidus,Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacteriumventriosum, Bacteroides fragilis ssp. fragilis, Coprococcus catus,Aerostipes hadrus, Eubacterium cylindroides, Eubacterium ruminantium,Staphylococcus epidermidis, Eubacterium limosum, Tissirella praeacuta,Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridiuminnocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcusflavefaciens, Bacteroides fragilis ssp. ovatus, Fusobacterium nucleatum,Fusobacterium mortiferum, Escherichia coli, Gemella morbillorum,Finegoldia magnus, Streptococcus intermedius, Ruminococcus lactaris,Eubacterium tenue, Eubacterium ramulus, Bacteroides clostridiiformisssp. clostridliformis, Bacteroides coagulans, Prevotella oralis,Prevotella ruminicola, Odoribacter splanchnicus, and Desuifomonas pigra.

In another aspect, a therapeutic composition comprises a fecalmicrobiota further supplemented, spiked, or enhanced with a fecalmicroorganism. In one aspect, a fecal microbiota is supplemented with anon-pathogenic (or with attenuated pathogenicity) bacterium ofClostridium, Collinsella, Dorea, Ruminococcus, Coprococcus, Prevotella,Veillonella, Bacteroides, Bacillus, or a combination thereof. In anotheraspect, a therapeutic composition comprises a fecal microbiota furthersupplemented, spiked, or enhanced with a species of Veillonellaceae,Firmicutes, Gammaproteobacteria, Bacteroidetes, or a combinationthereof. In another aspect, a therapeutic composition comprises a fecalmicrobiota further supplemented with fecal bacterial spores. In oneaspect, fecal bacterial spores are Clostridium spores, Bacillus spores,or both. In another aspect, a therapeutic composition comprises a fecalmicrobiota further supplemented, spiked, or enhanced with a Bacteroidesspecies selected from the group consisting of Bacteroides coprocola,Bacteroides plebeius, Bacteroides massiliensis, Bacteroides vulgatus,Bacteroides helcogenes, Bacteroides pyogenes, Bacteroides tectus,Bacteroides uniformis, Bacteroides stercoris, Bacteroides eggerthii,Bacteroides finegoldii, Bacteroides thetaiotaomicron, Bacteroidesovatus, Bacteroides acidifaciens, Bacteroides caccae, Bacteroidesnordii, Bacteroides salyersiae, Bacteroides fragilis, Bacteroidesintestinalis, Bacteroides coprosuis, Bacteroides distasonis, Bacteroidesgoldsteinii, Bacteroides merdae, Bacteroides forsythus, Bacteroidessplanchnicus, Bacteroides capillosus, Bacteroides cellulosolvens, andBacteroides ureolyticus.

In an aspect, a therapeutic composition comprises a fecal microbiotafrom a subject selected from the group consisting of a human, a bovine,a dairy calf, a ruminant, an ovine, a caprine, or a cervine. In anotheraspect, a therapeutic composition can be administered to a subjectselected from the group consisting of a human, a bovine, a dairy calf, aruminant, an ovine, a caprine, or a cervine. In an aspect, a therapeuticcomposition is substantially or nearly odourless.

In an aspect, a therapeutic composition provided here comprises a fecalmicrobiota preparation comprising a Shannon Diversity Index of greaterthan or equal to 0.3, greater than or equal to 0.4, greater than orequal to 0.5, greater than or equal to 0.6, greater than or equal to0.7, greater than or equal to 0.8, greater than or equal to 0.9, greaterthan or equal to 1.0, greater than or equal to 1.1, greater than orequal to 1.2, greater than or equal to 1.3, greater than or equal to1.4, greater than or equal to 1.5, greater than or equal to 1.6, greaterthan or equal to 1.7, greater than or equal to 1.8, greater than orequal to 1.9, greater than or equal to 2.0, greater than or equal to2.1, greater than or equal to 2.2, greater than or equal to 2.3, greaterthan or equal to 2.4, greater than or equal to 2.5, greater than orequal to 3.0, greater than or equal to 3.1, greater than or equal to3.2, greater than or equal to 3.3, greater than or equal to 3.4, greaterthan or equal to 3.5, greater than or equal to 3.6, greater than orequal to 3.7, greater than or equal to 3.8, greater than or equal to3.9, greater than or equal to 4.0, greater than or equal to 4.1, greaterthan or equal to 4.2, greater than or equal to 4.3, greater than orequal to 4.4, greater than or equal to 4.5, or greater than or equal to5.0. In another aspect, a therapeutic composition comprises fecalmicrobiota comprising a Shannon Diversity Index of between 0.1 and 3.0,between 0.1 and 2.5, between 0.1 and 2.4, between 0.1 and 2.3, between0.1 and 2.2, between 0.1 and 2.1, between 0.1 and 2.0, between 0.4 and2.5, between 0.4 and 3.0, between 0.5 and 5.0, between 0.7 and 5.0,between 0.9 and 5.0, between 1.1 and 5.0, between 1.3 and 5.0, between1.5 and 5.0, between 1.7 and 5.0, between 1.9 and 5.0, between 2.1 and5.0, between 2.3 and 5.0, between 2.5 and 5.0, between 2.7 and 5.0,between 2.9 and 5.0, between 3.1 and 5.0, between 3.3 and 5.0, between3.5 and 5.0, between 3.7 and 5.0, between 31.9 and 5.0, or between 4.1and 5.0. In one aspect, a Shannon Diversity Index is calculated at thephylum level. In another aspect, a Shannon Diversity Index is calculatedat the family level. In one aspect, a Shannon Diversity Index iscalculated at the genus level. In another aspect, a Shannon DiversityIndex is calculated at the species level. In a further aspect, atherapeutic composition comprises a preparation of flora in proportionalcontent that resembles a normal healthy human fecal flora.

In a further aspect, a therapeutic composition comprises fecal bacteriafrom at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different families. In anaspect, a therapeutic composition provided here comprises a fecalmicrobiota comprising a weight ratio between fecal-derived non-livingmaterial and fecal-derived biological material of no greater than 0.05%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or 10%. In another aspect, a therapeutic compositionprovided here comprises a fecal microbiota comprising a weight ratiobetween fecal-derived non-living material and fecal-derived biologicalmaterial of no greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95%. In another aspect, a therapeuticcomposition provided here comprises, consists of, or consistsessentially of, particles of non-living material and/or particles ofbiological material of a fecal sample that passes through a sieve, acolumn, or a similar filtering device having a sieve, exclusion, orparticle filter size of 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2mm. “Non-living material” does not include an excipient, e.g., apharmaceutically inactive substance, such as a cryoprotectant, added toa processed fecal material. “Biological material” refers to the livingmaterial in fecal material, and includes microbes including prokaryoticcells, such as bacteria and archaea (e.g., living prokaryotic cells andspores that can sporulate to become living prokaryotic cells),eukaryotic cells such as protozoa and fungi, and viruses. In one aspect,“biological material” refers to the living material, e.g., the microbes,eukaryotic cells, and viruses, which are present in the colon of anormal healthy human. In an aspect, a therapeutic composition providedor comprises an extract of human feces where the composition issubstantially odorless. In an aspect, a therapeutic composition providedor comprises fecal material or a fecal floral preparation in alyophilized, crude, semi-purified or purified formulation.

In an aspect, a fecal microbiota in a therapeutic composition compriseshighly refined or purified fecal flora, e.g., substantially free ofnon-floral fecal material. In an aspect, a fecal microbiota can befurther processed, e.g., to undergo microfiltration before, after, orbefore and after sieving. In another aspect, a highly purified fecalmicrobiota product is ultra-filtrated to remove large molecules butretain the therapeutic microflora, e.g., bacteria.

In another aspect, a fecal microbiota in a therapeutic composition usedherein comprises or consists essentially of a substantially isolated ora purified fecal flora or entire (or substantially entire) microbiotathat is (or comprises) an isolate of fecal flora that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%,99.8% or 99.9% isolated or pure, or having no more than about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% or more non-fecalfloral material; or, a substantially isolated, purified, orsubstantially entire microbiota as described in Sadowsky et al., WO2012/122478 A1, or as described in Borody et al., WO 2012/016287 A2. Inone aspect, a fecal microbiota preparation comprises a weight ratiobetween fecal-derived non-living material and fecal-derived biologicalmaterial of no greater than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40$, or 50%.

In an aspect, a fecal microbiota in a therapeutic composition comprisesa donor's substantially entire or non-selective fecal microbiota,reconstituted fecal material, or synthetic fecal material. In anotheraspect, the fecal microbiota in a therapeutic composition comprises noantibiotic resistant population. In another aspect, a therapeuticcomposition comprises a fecal microbiota and is largely free ofextraneous matter (e.g., non-living matter including acellular mattersuch as residual fiber, DNA, RNA, viral coat material, non-viablematerial; and living matter such as eukaryotic cells from the fecalmatter's donor).

In an aspect, a fecal microbiota in a therapeutic composition usedherein is derived from disease-screened fresh homologous feces orequivalent freeze-dried and reconstituted feces. In an aspect, a freshhomologous feces does not include an antibiotic resistant population. Inanother aspect, a fecal microbiota in a therapeutic composition isderived from a synthetic fecal composition. In an aspect, a syntheticfecal composition comprises a preparation of viable flora whichpreferably in proportional content, resembles normal healthy human fecalflora which does not include antibiotic resistant populations. Suitablemicroorganisms may be selected from the following: Bacteroides,Eubacterium, Fusobacterium, Propionibacterium, Lactobacillus,Ruminococcus, Escherichia coli, Gemmiger, Clostridium, Desulfomonas,Peptostreptococcus, Bifidobacterium, Collinsella, Coprococcus, Dorea,and Ruminococcus.

In an aspect, a therapeutic composition is combined with other adjuvantssuch as antacids to dampen bacterial inactivation in the stomach. (e.g.,Mylanta, Mucaine, Gastrogel). In another aspect, acid secretion in thestomach could also be pharmacologically suppressed using H2-antagonistsor proton pump inhibitors. An example H2-antagonist is ranitidine. Anexample proton pump inhibitor is omeprazole. In one aspect, an acidsuppressant is administered prior to administering, or inco-administration with, a therapeutic composition.

In an aspect, a therapeutic composition is administered in the form of:an enema composition which can be reconstituted with an appropriatediluent; enteric-coated capsules; enteric-coated microcapsules;acid-resistant tablet; acid-resistant capsules; acid-resistantmicrocapsules; powder for reconstitution with an appropriate diluent fornaso-enteric infusion or colonoscopic infusion; powder forreconstitution with appropriate diluent, flavoring and gastric acidsuppression agent for oral ingestion; powder for reconstitution withfood or drink; or food or food supplement comprising enteric-coatedand/or acid-resistant microcapsules of the composition, powder, jelly,or liquid.

In an aspect, a treatment method effects a cure, reduction of thesymptoms, or a percentage reduction of symptoms of a disorder (e.g.,ASD). The change of flora is preferably as “near-complete” as possibleand the flora is replaced by viable organisms which will crowd out anyremaining, original flora. Typically the change in enteric floracomprises introduction of an array of predetermined flora into thegastro-intestinal system, and thus in a preferred form the method oftreatment comprises substantially or completely displacing pathogenicenteric flora in patients requiring such treatment.

In another aspect, a therapeutic composition can be provided togetherwith a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” refers to a non-toxic solvent,dispersant, excipient, adjuvant, or other material which is mixed with alive bacterium in order to permit the formation of a pharmaceuticalcomposition, e.g., a dosage form capable of administration to thepatient. A pharmaceutically acceptable carrier can be liquid (e.g.,saline), gel or solid form of diluents, adjuvant, excipients or an acidresistant encapsulated ingredient. Suitable diluents and excipientsinclude pharmaceutical grades of physiological saline, dextrose,glycerol, mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, cellulose, magnesium carbonate, and the like, andcombinations thereof. In another aspect, a therapeutic composition maycontain auxiliary substances such as wetting or emulsifying agents,stabilizing or pH buffering agents. In an aspect, a therapeuticcomposition contains about 1%-5%, 5%-10%, 10%-15%, 15-20%, 20%-25%,25-30%, 30-35%, 40-45%, 50%-55%, 1%-95%, 2%-95%, 5%-95%, 10%-95%,15%-95%, 20%-95%, 25%-95%, 30%-95%, 35%-95%, 40%-95%, 45%-95%, 50%-95%,55%-95%, 60%-95%, 65%-95%, 70%-95%, 45%-95%, 80%-95%, or 85%-95% ofactive ingredient. In an aspect, a therapeutic composition containsabout 2%-70%, 5%-60%, 10%-50%, 15%-40%, 20%-30%, 25%-60%, 30%-60%, or35%-60% of active ingredient.

In an aspect, a therapeutic composition can be incorporated intotablets, drenches, boluses, capsules or premixes. Formulation of theseactive ingredients into such dosage forms can be accomplished by meansof methods well known in the pharmaceutical formulation arts. See, e.g.,U.S. Pat. No. 4,394,377. Filling gelatin capsules with any desired formof the active ingredients readily produces capsules. If desired, thesematerials can be diluted with an inert powdered diluent, such as sugar,starch, powdered milk, purified crystalline cellulose, or the like toincrease the volume for convenience of filling capsules.

In an aspect, conventional formulation processes can be used to preparetablets containing a therapeutic composition. In addition to the activeingredients, tablets may contain a base, a disintegrator, an absorbent,a binder, and a lubricant. Typical bases include lactose, sugar, sodiumchloride, starch and mannitol. Starch is also a good disintegrator as isalginic acid. Surface-active agents such as sodium lauryl sulfate anddioctyl sodium sulphosuccinate are also sometimes used. Commonly usedabsorbents include starch and lactose. Magnesium carbonate is alsouseful for oily substances. As a binder there can be used, for example,gelatin, gums, starch, dextrin, polyvinyl pyrrolidone and variouscellulose derivatives. Among the commonly used lubricants are magnesiumstearate, talc, paraffin wax, various metallic soaps, and polyethyleneglycol.

In an aspect, for preparing solid compositions such as tablets, anactive ingredient is mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, or other pharmaceutical diluents, e.g. water, to forma solid preformulation composition containing a homogeneous mixture of acomposition of the present disclosure. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulationcomposition is then subdivided into unit dosage forms of the typedescribed above containing a desired amount of an active ingredient(e.g., at least about 10⁵, 10⁶, 10⁷, 10 ⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cfu). A therapeutic composition used herein can be flavored.

In an aspect, a therapeutic composition can be a tablet or a pill. Inone aspect, a tablet or a pill can be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, a tablet or pill can comprise an inner dosage and an outerdosage component, the latter being in the form of an envelope over theformer. The two components can be separated by an enteric layer whichserves to resist disintegration in the stomach and permits the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

In an aspect, a therapeutic composition is formulated as a delayed orgradual enteric release form. In an aspect, a delayed or gradual entericrelease formulation comprises the use of cellulose acetate, polyethyleneglycerol, or both. In an aspect, a delayed or gradual enteric releaseformulation comprises the use of a hydroxypropylmethylcellulose (HPMC),a microcrystalline cellulose (MCC), magnesium stearate, or a combinationthereof. In an aspect, a delayed or gradual enteric release formulationcomprises the use of a poly(meth)acrylate, a methacrylic acid copolymerB, a methyl methacrylate, a methacrylic acid ester, apolyvinylpyrrolidone (PVP), a PVP-K90, or a combination thereof. In anaspect, a delayed or gradual enteric release formulation comprises theuse of a solid inner layer sandwiched between two outer layers; whereinthe solid inner layer comprises the pharmaceutical composition andanother component selected from the group consisting of a disintegrant,an exploding agent, an effervescent or any combination thereof; whereinthe outer layer comprises a substantially water soluble, a crystallinepolymer, or both. In an aspect, a delayed or gradual enteric releaseformulation comprises the use of a non-swellable diffusion matrix.

In another aspect, a delayed or gradual enteric release formulationcomprises the use of a bilayer tablet or capsule which comprises a firstlayer comprising a polyalkylene oxide, a polyvinylpyrrolidone, alubricant, or a mixture thereof, and a second osmotic push layercomprising polyethylene oxide, carboxy-methylcellulose, or both. In anaspect, a delayed or gradual enteric release formulation comprises theuse of a release-retarding matrix material selected from the groupconsisting of an acrylic polymer, a cellulose, a wax, a fatty acid,shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil,polyvinylpyrrolidine, a vinyl acetate copolymer, a vinyl alcoholcopolymer, polyethylene oxide, an acrylic acid and methacrylic acidcopolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylatepolymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylatecopolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacrylicacid alkylamide copolymer, a poly(methyl methacrylate), apoly(methacrylic acid anhydride), a methyl methacrylate polymer, apolymethacrylate, a poly(methyl methacrylate) copolymer, apolyacrylamide, an aminoalkyl methacrylate copolymer, a glycidylmethacrylate copolymer, a methyl cellulose, an ethylcellulose, acarboxymethylcellulose, a hydroxypropylmethylcellulose, a hydroxymethylcellulose, a hydroxyethyl cellulose, a hydroxypropyl cellulose, acrosslinked sodium carboxymethylcellulose, a crosslinkedhydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol,a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenatedfat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax,glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolicacid, a co-polymer of lactic and glycolic acid, carboxymethyl starch,potassium methacrylate/divinylbenzene copolymer, crosslinkedpolyvinylpyrrolidone, poly inylalcohols, polyvinylalcohol copolymers,polyethylene glycols, non-crosslinked polyvinylpyrrolidone, polyvinylacetates, polyvinylacetate copolymers, or any combination thereof. In anaspect, a delayed or gradual enteric release formulation comprises theuse of a microenvironment pH modifier.

In an aspect, a therapeutic composition can be a drench. In one aspect,a drench is prepared by choosing a saline-suspended form of atherapeutic composition. A water-soluble form of one ingredient can beused in conjunction with a water-insoluble form of the other bypreparing a suspension of one with an aqueous solution of the other.Water-insoluble forms of either active ingredient may be prepared as asuspension or in some physiologically acceptable solvent such aspolyethylene glycol. Suspensions of water-insoluble forms of eitheractive ingredient can be prepared in oils such as peanut, corn, sesameoil or the like; in a glycol such as propylene glycol or a polyethyleneglycol; or in water depending on the solubility of a particular activeingredient. Suitable physiologically acceptable adjuvants may benecessary in order to keep the active ingredients suspended. Adjuvantscan include and be chosen from among the thickeners, such ascarboxymethylcellulose, polyvinyl pyrrolidone, gelatin and thealginates. Surfactants generally will serve to suspend the activeingredients, particularly the fat-soluble propionate-enhancingcompounds. Most useful for making suspensions in liquid nonsolvents arealkylphenol polyethylene oxide adducts, naphthalenesulfonates,alkylbenzene-sulfonates, and the polyoxyethylene sorbitan esters. Inaddition many substances, which affect the hydrophilicity, density andsurface tension of the liquid, can assist in making suspensions inindividual cases. For example, silicone anti-foams, glycols, sorbitol,and sugars can be useful suspending agents.

In an aspect, a therapeutic composition comprises non-pathogenic sporesof one or more, two or more, three or more, or four or more Clostridiumspecies selected from the group consisting of Clostridium absonum,Clostridium argentinense, Clostridium baratii, Clostridium botulinum,Clostridium cadaveris, Clostridium carnis, Clostridium celatum,Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium fallax, Clostridium felsineum, Clostridiumghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridiumhastiforme, Clostridium histolyticum, Clostridium indolis, Clostridiumirregulare, Clostridium limosum, Clostridium malenominatum, Clostridiumnovyi, Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium sardiniense, Clostridiumsartagoforme, Clostridium scindens, Clostridium septicum, Clostridiumsordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridiumsporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridiumtertium, Clostridium tetani, Clostridium welchii, and Clostridiumvillosum. In an aspect, a therapeutic composition comprises one or more,two or more, three or more, or four or more non-pathogenic Bacteroidesspecies selected from the group of Bacteroides coprocola, Bacteroidesplebeius, Bacteroides massiliensis, Bacteroides vulgatus, Bacteroideshelcogenes, Bacteroides pyogenes, Bacteroides tectus, Bacteroidesuniformis, Bacteroides stercoris, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides thetaiotaomicron, Bacteroides ovatus,Bacteroides acidifaciens, Bacteroides caccae, Bacteroides nordii,Bacteroides salyersiae, Bacteroides fragilis, Bacteroides intestinalis,Bacteroides coprosuis, Bacteroides distasonis, Bacteroides goldsteinii,Bacteroides merdae, Bacteroides forsythus, Bacteroides splanchnicus,Bacteroides capillosus, Bacteroides cellulosolvens, and Bacteroidesureolyticus. The foregoing Clostridium and Bacteroides can be eithercultured or purified and can be used in combination in a singlecombination for a synergistic effect.

In an aspect, a therapeutic composition comprises purified, isolated, orcultured viable non-pathogenic Clostridium and a plurality of purified,isolated, or cultured viable non-pathogenic microorganisms from one ormore genera selected from the group consisting of Collinsella,Coprococcus, Dorea, Eubacterium, and Ruminococcus. In another aspect, atherapeutic composition comprises a plurality of purified, isolated, orcultured viable non-pathogenic microorganisms from one or more generaselected from the group consisting of Clostridium, Collinsella,Coprococcus, Dorea, Eubacterium, and Ruminococcus.

In an aspect, a therapeutic composition comprises two or more generaselected from the group consisting of Collinsella, Coprococcus, Dorea,Eubacterium, and Ruminococcus. In another aspect, a therapeuticcomposition comprises two or more genera selected from the groupconsisting of Coprococcus, Dorea, Eubacterium, and Ruminococcus. In afurther aspect, a therapeutic composition comprises one or more, two ormore, three or more, four or more, or five or more species selected fromthe group consisting of Coprococcus catus, Coprococcus comes, Dorealongicatena, Eubacterium eligens, Eubacterium hadrum, Eubacteriumhallii, Eubacterium rectale, and Ruminococcus torques.

In one aspect, a pharmaceutical composition is in an anaerobic packageor container. In another aspect, a pharmaceutical composition furthercomprises an oxygen scavenger. In one aspect, a container can be madeoxygen free by e.g., incorporating into the container a built in orclipped-on oxygen-scavenging mechanism, e.g., oxygen scavenging pelletsas described e.g., in U.S. Pat. No. 7,541,091. In another aspect, thecontainer itself is made of an oxygen scavenging material, e.g., oxygenscavenging iron, e.g., as described by O2BLOCK™, or equivalents, whichuses a purified and modified layered clay as a performance-enhancingcarrier of oxygen-scavenging iron; the active iron is dispersed directlyin the polymer. In one aspect, oxygen-scavenging polymers are used tomake the container itself or to coat the container, or as pellets to beadded; e.g., as described in U.S. Pat. App. Pub. 20110045222, describingpolymer blends having one or more unsaturated olefinic homopolymers orcopolymers; one or more polyamide homopolymers or copolymers; one ormore polyethylene terephthalate homopolymers or copolymers; that exhibitoxygen-scavenging activity. In one aspect, oxygen-scavenging polymersare used to make the container itself or to coat the container, or aspellets to be added; e.g., as described in U.S. Pat. App. Pub.20110008554, describing compositions comprising a polyester, acopolyester ether and an oxidation catalyst, wherein the copolyesterether comprises a polyether segment comprisingpoly(tetramethylene-co-alkylene ether). In one aspect, oxygen-scavengingpolymers are used to make the container itself or to coat the container,or as pellets to be added; e.g., as described in U.S. Pat. App. Pub.201000255231, describing a dispersed iron/salt particle in a polymermatrix, and an oxygen scavenging film with oxygen scavengingparticulates.

In preferred aspects, purified fecal microbiota is obtained from acarefully screened, healthy, neurotypical human donor. Microbiota isseparated from fecal material collected from healthy donors, mixed witha cryopreservative, stored as a frozen liquid suspension with thecryopreservative, and thawed prior to administration in liquid form.Based on the route of administration, the purified fecal microbiota canbe provided as fresh, frozen-thawed, or lyophilized live microbiota. Insome cases, purified fecal microbiota is administered to a human subjectin the form of an oral dose. In other cases, purified fecal microbiotais administered in the form of a rectal dose.

In some cases, the dosage form comprises any suitable form of livemicrobiota (fresh, frozen, lyophilized, etc.) and is formulated foradministration to a human subject orally, by nasogastric tube, bycolonoscopy, or anally. In some cases, the dosage is administered as asolution. In other cases, the dosage is administered as solid dosageforms such as, for example, capsules, tablets, powders, and granules. Insuch solid dosage forms, purified fecal microbiota is admixed with atleast one inert excipient (or carrier), a filler or extender (e.g.,starches, lactose, sucrose, mannitol, or silicic acid), a binder (e.g.,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, or acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, sodium carbonate), an absorption accelerators,a wetting agent (e.g., cetyl alcohol or glycerol monostearate), anadsorbent (e.g., kaolin or bentonite), and/or a lubricant (e.g., talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, or mixtures thereof). In the case of capsules andtablets, the dosage forms may also comprise buffering agents.

A tablet comprising purified fecal microbiota can, for example, be madeby compressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquidtomoisten the mixture. In exemplary aspects, the dosage form comprises apowder prepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from purified, frozen fecal microbiota atextremely low pressures.

The specific dosage and dosage range that can be used depends on anumber of factors, and the determination of dosage ranges and optimaldosages for a particular patient is well within the ordinary skill ofone in the art in view of this disclosure. It is further understood,however, that the specific dose level for any particular human willdepend upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, gender, anddiet of the human, the time of administration, the route ofadministration, the rate of excretion, any drug combination, and theseverity of any disorder being treated.

In exemplary aspect, purified fecal microbiota is administered to asubject in multiple doses. For example, purified fecal microbiota can beadministered to a subject according to a method provided herein inmultiple doses over a time period of about two days to about eightweeks.

Prior to administration of purified fecal microbiota, any suitableantibiotic can be administered to the subject. In exemplary aspects, theantibiotic is a non-absorbed or minimally-absorbed antibiotic such as,for example, vancomycin or rifaximin. Antibiotics are administered tothe subject via any appropriate delivery route. One of skill in the artcan develop appropriate dose delivery methods. Preferably, theantibiotic is administered to the subject orally. In another aspect, anASD treatment method requires no antibiotic pretreatment. In a furtheraspect, an ASD treatment method requires no bowel preparation or bowelcleansing. In another aspect, an ASD treatment method requires neitherantibiotic pretreatment nor bowel cleansing prior to administering apharmaceutical composition comprising a fecal microbiota preparation.

In some cases, the antibiotic is administered in multiple doses before abowel cleanse is performed. In some cases, administration of theantibiotic is initiated at least seven days (e.g., at least 7, 9, 10,12, 14, 18, or 21 days) before the bowel cleanse. In preferred aspects,the bowel cleanse is preceded by fasting of the human subject.

Following administration of an antibiotic, the subject undergoes a bowelcleanse. In exemplary aspects, the bowel cleanse comprises administeringto the subject a product such as MoviPrep®, a commercial bowel prep forcolonoscopy. Preferably, the bowel cleanse removes residual vancomycinand cleanses the lower gastrointestinal tract.

In exemplary aspects, the method further comprises administering to thesubject a stomach acid suppressant. Stomach acid suppressants, alsoknown as gastric acid suppressants, suitable for use according to amethod provided herein include, without limitation, proton pumpinhibitors (PPIs) and histamine blockers. In some cases, the stomachacid suppressant is Prilosec and is administered to the subject one ormore days in advance of oral administration of purified fecalmicrobiota. In some cases, the stomach acid suppressant is administeredone week prior to oral administration of purified fecal microbiota.

In another aspect, provided herein are unit dosage forms comprisingpurified fecal microbiota. In some cases, unit dosage forms describedherein are provided as part of a kit. Such a kit could include apurified fecal microbiota dosage and, optionally, a delivery device toadminister the composition to the subject or instructions foradministering the dosage to a subject via an appropriate delivery route.In some cases, the dosage form comprises any suitable form of livemicrobiota (fresh, frozen, lyophilized, etc.) and is formulated foradministration to a human subject orally, by nasogastric tube, bycolonoscopy, or anally. As described herein, dosage forms suitable forkits provided herein include, without limitation, liquid solutions,capsules, tablets, powders, granules, and lyophilized forms.

In a further aspect, provided herein is use of a purified compositionfor manufacture of a medicament for treating autism spectrum disorder orfor reducing the severity of one or more symptoms of autism spectrumdisorder.

It will be appreciated that compositions, dosage forms, and medicamentsas described herein include combination pharmaceutical compositions inwhich one or more additional compounds or medications are added to orotherwise co-administered with a purified fecal microbiota composition.

Articles “a” and “an” are used herein to refer to one or to more thanone (i.e., at least one) of the grammatical object of the article. Byway of example, “an element” means at least one element and can includemore than one element.

The following paragraphs list a subset of exemplary embodiments.

Embodiment 1

A method of treating an autism spectrum disorder in a human subject,comprising:

-   -   (a) administering an antibiotic to a human subject;    -   (b) subjecting the human subject to a bowel cleanse; and    -   (c) administering purified fecal microbiota to the human        subject; wherein an autism spectrum disorder is treated in the        human subject.

Embodiment 2

The method of Embodiment 1, wherein the antibiotic is a non-absorbableantibiotic orally-administered to the human subject.

Embodiment 3

The method of Embodiment 1 or 2, wherein the antibiotic is vancomycin.

Embodiment 4

The method of any one of preceding Embodiments 1-3, wherein theantibiotic is administered in multiple doses before the bowel cleanse.

Embodiment 5

The method of any one of preceding Embodiments 1-4, whereinadministration of the antibiotic is initiated at least seven days beforethe bowel cleanse.

Embodiment 6

The method of any one of preceding Embodiments 1-4, whereinadministration of the antibiotic is initiated at least fourteen daysbefore the bowel cleanse.

Embodiment 7

The method of any one of preceding Embodiments 1-6, wherein the bowelcleanse is preceded by fasting of the human subject.

Embodiment 8

The method of any one of preceding Embodiments 1-7, wherein the purifiedfecal microbiota is obtained from a neurotypical human donor.

Embodiment 9

The method of any one of preceding Embodiments 1-8, wherein the purifiedfecal microbiota is administered to the human subject in the form of anoral dose.

Embodiment 10

The method of Embodiment 9, wherein the method includes a step ofadministering an acid suppressant prior to administration of thepurified fecal microbiota.

Embodiment 11

The method of any one of preceding Embodiments 1-8, wherein the purifiedfecal microbiota is administered to the human subject in the form of arectal dose.

Embodiment 12

The method of any one of preceding Embodiments 1-11, wherein thepurified fecal microbiota is administered in multiple doses.

Embodiment 13

The method of any one of preceding Embodiments 12, wherein the purifiedfecal microbiota is administered in multiple doses over a time period ofabout two days to about eight weeks.

Embodiment 14

The method of any one of preceding Embodiments 1-13, wherein thepurified fecal microbiota is in the form of fresh, frozen-thawed, orlyophilized live microbiota.

Embodiment 15

The method of any one of preceding Embodiments 1-14, wherein the humansubject exhibits a significant reduction in autism symptom severity asassessed by the Childhood Autism Rating Scale (CARS) after said methodas compared to before initiating the method.

Embodiment 16

The method of any one of preceding Embodiments 1-14, wherein the humansubject exhibits at least a 10% reduction in autism symptom severity asassessed by the Childhood Autism Rating Scale (CARS) after said methodas compared to before initiating the method.

Embodiment 17

The method of any one of preceding Embodiments 1-14, wherein the humansubject exhibits at least a 20% reduction in autism symptom severity asassessed by the Childhood Autism Rating Scale (CARS) after said methodas compared to before initiating the method.

Embodiment 18

The method of any one of preceding Embodiments 1-17, wherein the humansubject treated by said method is characterized by significantly fewerspecies of gut bacteria before said method of treatment as compared to aneurotypical human.

Embodiment 19

The method of any one of preceding Embodiments 1-17, wherein the humansubject treated by said method is characterized by about 20% fewerspecies of gut bacteria before said method of treatment as compared to aneurotypical human.

Embodiment 20

The method of any one of preceding Embodiments 1-19, wherein the humansubject does not present gastrointestinal distress symptoms prior toinitiating said method.

Embodiment 21

A method of reducing severity of an autism spectrum disorder in a humansubject, comprising:

-   -   (a) orally-administering a non-absorbable antibiotic to an        autistic human subject;    -   (b) subjecting the autistic human subject to a bowel cleanse;        and    -   (c) administering purified fecal microbiota from a neurotypical        human donor to the human subject; wherein the human subject        exhibits a significant reduction in symptom severity as assessed        by the Childhood Autism Rating Scale (CARS) after said method as        compared to before initiating the method.

Embodiment 22

The method of Embodiment 21, wherein the human subject exhibits at leasta 10% reduction in autism symptom severity as assessed by the ChildhoodAutism Rating Scale (CARS) after said method as compared to beforeinitiating the method.

Embodiment 23

The method of Embodiment 21, wherein the human subject exhibits at leasta 20% reduction in autism symptom severity as assessed by the ChildhoodAutism Rating Scale (CARS) after said method as compared to beforeinitiating the method.

Embodiment 24

The method of any one of Embodiments 20-22, wherein the human subjectdoes not present gastrointestinal distress symptoms prior to initiatingsaid method.

Embodiment 25

A purified fecal microbiota dosage for use in treating an autismspectrum disorder in a human subject according to the method of any oneof Embodiments 1-20.

Embodiment 26

A purified fecal microbiota dosage for use in reducing severity of anautism spectrum disorder symptom in an human subject according to themethod of any one of Embodiments 21-24.

The disclosure may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of thedisclosure. The following examples are presented in order to more fullyillustrate the preferred aspects of the disclosure and should in no waybe construed, however, as limiting the broad scope of the disclosure.Therefore, the scope of the appended claims should not be limited to thedescription of the aspects contained herein.

EXAMPLES Example 1: Treating Autistic Children Using Microbiota TransferTherapy (MTT)

The FDA and ASU's Human Subject Board approved a pilot study of 20autistic children, ages 7-17, to participate in a trial evaluating thesafety and tolerability of a fecal microbiota-based treatment designedto reduce the symptoms of autism by improving the gastrointestinalmicrobiota function. As described herein, this treatment includedtransfer of purified gut bacteria from a healthy person to childrendiagnosed as having autism spectrum disorder.

The general study design was an open-label clinical trial involving 18children (ages 7-17 years) with ASD who were diagnosed by the AutismDiagnostic Interview-Revised (ADI-R) and had moderate to severegastrointestinal problems. Each child participated in the study for 18weeks in total, a 10 week treatment and a follow-up 8 week observationperiod after the treatment stopped. For the fecal material transplant(FMT) treatment, we compared two routes of administration, oral versusrectal, for the initial dose, followed by a lower maintenance dosagegiven orally for 7-8 weeks.

The protocol was approved by FDA (Investigational new drug number 15886)and the Institutional Review Board of Arizona State University (ASU IRBProtocol #: 00001053). The study was advertised by email toapproximately 2500 ASD families in Arizona, using the contact list ofthe Autism Society of Greater Phoenix and the Autism/Asperger's ResearchProgram at Arizona State University. Families with children who met thestudy inclusion and exclusion criteria had a 1-hour individual phonecall to discuss the study. After the phone call, families who signed theparent permission form and child assent form were provided with initialquestionnaires to complete. We also sent them a letter for theirpersonal physician to double-check their medications and for thephysician to be aware of the delivery of the vancomycin, Prilosec, andthe fecal transplant

Beneficial bacteria (a non-selective fecal microbiota preparation) wereprepared from human donor stools. Fecal samples were collected fromcarefully-screened healthy donors (90% of general population rejected)and purified extensively to retain only bacteria. Specifically, themicrobiota was separated from fecal material collected from carefullyscreened, healthy donors, stored in a cryopreservative in a frozenliquid suspension with a cryopreservative, and thawed prior toadministration in liquid form. Each purified sample of beneficialbacteria contained 1000 or more bacterial species. By comparison,standard commercially available probiotics include 1 to 10 bacterialspecies.

Example 2: Subject Recruitment

The study began with a verification of an autism spectrum diagnosisusing the Autism Diagnostic Interview-Revised (ADI-R), which involved aphone interview of the parents with our ADI-R evaluator. The studyphysician assessed general physical health through an initial 30 minutemeeting with participants and an extensive review of the participants'last 2 years of medical records and height/weight/growth charts in orderto check for exclusion criteria. Participant exclusion criteria includeantibiotics in last 6 months and probiotics in last 3 months,single-gene disorder, major brain malformation, tube feeding, severe GIproblems that require immediate treatment (life-threatening), UlcerativeColitis, Crohn's disease, diagnosed Celiac Disease, EosinophilicGasteroenteritis, severely underweight/malnourished, andrecent/scheduled surgeries. None of the neurotypical children wasdiagnosed with mental disorders including ASD, ADHD, depression, andanxiety, and neurotypical children did not have first-degree relativesof individuals with ASD. From participants, we collected initial blood,urine, and stool samples and parents were asked to fill in diet diariesof their child for one week at the beginning of the study. Participantswere recruited primarily from the greater Phoenix, Ariz. area, but threeparticipants were from outside that area. Neurotypical families wererecruited from friends of the ASD families and professionals who workwith ASD families.

Example 3: Trial Participants

Eighteen autism participants (each from a different family) ages 7-17years with moderate to severe GI problems and moderate to high cognitivefunctioning. Twenty participants were recruited into the study, but twodid not enter the treatment phase of the study before the treatmentstarted. One participant was disqualified due to a change in medication,and one decided not to participate. Characteristics of 18 studyparticipants and their medical history are listed in Table 2. All 18participants that entered the treatment phase completed the 19-weektrial. The post-treatment data presented herein were collected for 13 ofthese 18 participants. In addition, 20 age- and gender-matchedneurotypical children from 13 families (6 families had 1 neurotypicalparticipant, and 7 families had 2 neurotypical participants) are alsorecruited. These 20 neurotypical children were monitored for 18 weeksbut not treated.

TABLE 2 Characteristics of study participants and their medical history.Children Neurotypical with ASD children p-value Total Number 18 20Male/Female 16/2 18/2 Age 11.0 +/− 2.7 11.1 +/− 2.5 n.s. BMI 19.9 +/−5.4 18.1 +/− 3.4 n.s. GSRS 4-point scale 28.1 +/− 4.3 18.8 +/− 4.0 P <0.001 (sum of all 15 items, minimum score for no symptoms is 15) Born byC-Section 61% 16% P < 0.01 Number of months 3.3 +/− 3.9  9.3 +/− 7.8 P <0.01 of breastfeeding exclusively (no formula) % using non-standard 39% 8% P < 0.05 formula (soy or other) Food allergy (moderate 56%  5% P <0.01 or severe) Other allergies (moderate 44% 10% P < 0.01 or severe)Eczema 56%  5% P < 0.01 Fiber consumption-child 8.9 +/− 4.3 11.8 +/− 4.9P = 0.07 Fiber consumption-mother 6.7 +/− 3.9 10.5 +/− 4.5 P = 0.02 Oralantibiotic use during 4.6 +/− 5.2  4.1 +/− 6.0 n.s. first 4 years oflife (number of rounds)

Example 4: Trial Protocol

The participants were given oral vancomycin (a non-absorbable broadspectrum antibiotic that stays in the GI tract) for 2 weeks to reducelevels of pathogenic bacteria, and then 1 day of low-volume colonoscopyprep MoviPrep® (a drink that flushes the bowels, to remove mostremaining gut bacteria and vancomycin) to clear the residual vancomycinand feces. The vancomycin was intended to kill off harmful bacteria, thefasting was intended to remove any remaining bacteria and to minimizeother luminal fecal material, and the colon cleanse helped remove thevancomycin and cleanse the lower GI Tract.

Following vancomycin treatment and bowel cleanse, participants receivedeither 2 days of high dose oral Microbiota Transfer Therapy (MTT, mixedin a chocolate milk, milk substitute, or juice) (dosage of 2.5×10¹² CFUper day) or a single dose of rectal MTT (dosage of 2.5×10¹² CFU for onegiven similar to an enema). The rectal dose was administered under thedirect supervision of the study physician, and the first oral dose wassimilarly administered in the presence of the physician. Participantswere randomly assigned to either the oral or rectal route ofadministration. If one administration route was not tolerated, or if thefamily preferred the other route, then participants had the option oftrying the other route. For the participants with initial oral dose, alower oral maintenance dose (2.5×10⁹ CFU) was followed for 8 weeks rightafter the major oral initial dose. Whereas, the major rectal initialdose was followed by waiting period of 1 week followed by a lower oralmaintenance dose (2.5×10⁹ CFU) for 7 weeks. The maintenance SHGM dosewere self-administered orally every day up to week 10. After treatmentwas stopped, participants were monitored for another 8 weeks.

Prilosec (omeprazole) was administered daily to reduce stomach acid andthereby increase viability of the MTT, starting on the 12th day of oralvancomycin treatment and continuing until the end of the maintenancedose. Table 3 provides a general treatment timeline.

TABLE 3 MTT Treatment Timeline Summary. Time Initial oral Initial rectal(Day) administration administration Day 1-14 Vancomycin* Day 12-74Prilosec* Day 15 MoviPrep* Day 16 Major oral dose Major rectal of MTT**dose of MTT** Day 17 Major oral dose of MTT — Day 18-24 Lowermaintenance oral dose of MTT — Day 25-74 Lower maintenance oral dose ofMTT*** Day 75-130 No treatment, observation period *Vancomycin: 40 mg/kgP.O. per day, divided into three doses, not to exceed 2 gm per day;Prilosec: 20 mg PO QD; MoviPrep: Standard kit was used with half thedosage being administered at approximately 10 am and the other half at 4pm on day fifteen only, to cleanse the bowel of vancomycin and feces.The dosage varies proportionately based on the body mass. **Initial oralroute: The dosage for the first 2 days will be 8.3 × 10¹¹ cells, t.i.d,for a total daily dose of 2.5 × 10¹² cells/day, for Day 16 and 17 only;Initial rectal route: 2.5 × 10¹² cells, 1x (Day 16 only) ***Maintenancedose: 2.5 × 10⁹ cells, 1x/day P.O.

Example 5: Fecal Microbiota Preparation Used for MTT

A human microbiota preparation, which comprises a highly purifiedstandardized extract from human feces (also called Standardized HumanGut Microbiota (SHGM)) was used. This is a full-spectrum product,containing all the bacteria present in the gut of very healthy donors.First, donors were carefully screened using an extensive healthquestionnaire and extensive medical testing to ensure optimal GI andoverall health; the screening process is so rigorous that 90% of donorsare eliminated, leaving only the 10% healthiest portion of thepopulation. The donated material is then extensively filtered andstandardized, following FDA Good Manufacturing Processes (GMP). Thefinal product is liquid form which can be frozen, and was proven to behighly effective for treating C. difficile (Hamilton et al., Am JGastroenterol. 2012 May; 107(5):761-7). The SHGM was stored in −80° C.freezers and then delivered to families on dry ice every week during thestudy. Families were instructed to keep the SHGM in a container with dryice, and thaw it shortly before use.

Two different doses of SHGM were used; the high major dose and a lowermaintenance dose. The high-dose SHGM was at a daily dosage of 2.5×10¹²cells. The rationale for two days of high dose was that after theMoviPrep and 1-day fast is presumably the most critical time in which toprovide new beneficial bacteria. The low-dose SHGM was at a dosage of2.5×10⁹ cells.

Example 6: Toleration of Study Medications

Vancomycin: The vancomycin was associated with two types of minoradverse events. One child developed an allergic rash upon administrationof oral vancomycin, but they were switched to vancomycin without orangeflavoring and the rash disappeared. Twelve of the 18 children had abehavioral reaction to the vancomycin, starting 1-4 days after the startof the vancomycin, and lasting 1-3 days in most cases, although 1participant had symptoms lasting for 3 weeks. In 7 cases, the symptomswere mild to moderate increase in hyperactivity, and in 5 cases thesymptoms were mild to moderate increase in tantrumming/aggression. Afterthese behavioral symptoms disappeared, GI symptoms and autism symptomsbegan improving. Similar results were reported in a previous study(Sandler, 2000), and parents of the study subjected had been informed toexpect this. The reaction may be due to release of bacterial toxins asthe vancomycin kills off harmful bacteria.

Prilosec: This was generally well-tolerated.

MoviPrep®: Many children had difficulty consuming this medication due totaste.

Rectal administration of Microbiota Transfer Therapy (MTT): This wassurprisingly well-tolerated by 6 of 6 recipients.

Oral administration of high-dose MTT: This was well-tolerated by 12 of13 recipients, but 1 participant experienced vomiting and was switchedto the rectal route.

Oral administration of maintenance dose MTT: This was well-tolerated byall participants.

CBC/ChemPanel: There were no major concerns regarding changes inComplete Blood Count (CBC) or blood chemistry panel (CBC). The followingminor changes were observed. There was a 5% decrease in potassium(p=0.01) from beginning to end of treatment, but all levels remain inthe normal range. After the vancomycin (2nd week of study), there was a8% increase in platelets (p=0.03). Four subjects had elevated levels atstart, and only 2 had elevated levels after vancomycin. There was a 26%drop in blood urea nitrogen (BUN) (p=0.002), but all stayed in normalrange. There was a 6% increase in albumin to globulin (A/G) ratio(p=0.03), with 1 slightly elevated. There was a 17% increase inaspartate amino transferase (AST) (p=0.01), but all remained in normalrange. There was a 24% increase in alanine amino transferase (ALT)(p=0.003), where 1 remained elevated and 2 became slightly elevated. Allof these values (platelets, BUN, A/G, AST, ALT) returned to similar tobaseline at the 3rd and 4th tests. Slight changes (1-2%) in red bloodcell indices (Mean corpuscular volume (MCV), Mean corpuscular hemoglobin(MCH), Mean corpuscular hemoglobin concentration (MCHC), and Red celldistribution width (RDW)) were observed.

Example 7: Adverse Effects

Children with ASD experienced temporary adverse effects at the beginningof vancomycin treatment. As listed in Table 4, one participant among the18 children with ASD (5%) developed an extensive rash, but the rashdisappeared when vancomycin was switched from a natural orange flavor toan unflavored form. Within 1-4 days after the start of the vancomycin,12 children with ASD had a temporary behavioral reaction to thevancomycin either involving hyperactivity (7 out of 12 cases; 39%) orTantrums/Aggression (5 out of 12 cases; 28%). The symptoms lasted 1-3days in most cases, except for one participant that had symptoms lastingfor 3 weeks. After the symptoms disappeared, GI symptoms and behavioralsymptoms began improving, which is similar to what Sandler et al.,Journal of Child Neurology 15, 429-35, (2000) reported in their oralvancomycin therapy for children with autism. Only one participant didnot tolerate the initial high-dose oral SHGM (nausea/vomiting) and wasswitched to initial rectal administration.

TABLE 4 Adverse effects. Adverse effect % adverse effects Rash 5% (dueto natural orange flavor in vancomycin) Hyperactivity 39%* (temporary:start of vancomycin only) Tantrums/Aggression 28%* (temporary: start ofvancomycin only) Nausea/vomiting 5% (due to high-dose SHGM) *Theseverity of symptoms ranged from mild to moderate.

Example 8: Assessments of Gastrointestinal Symptoms

Gastrointestinal Symptom Rating Scale (GSRS) is an assessment of GIsymptoms during the previous week, based on 15 questions, which are thenscored in 5 domains: Abdominal Pain, Reflux, Indigestion, Diarrhea, andConstipation. We report a score for each domain based on the averagewithin the questions in that domain. The original GSRS used a 4-pointscale, but we used a revised version which included 7-point Likert scalewhich also has simpler language. The GSRS was assessed on days 0, 7, 14,21, 28, 35, 42, 56, 74, and 130. One of ordinary skill in the artunderstands that GSRS is only one way to assess GI symptoms. Othersimilar tools can be used or designed to evaluate GI symptoms.

Daily Stool Records (DSR) were collected at baseline for two weeks,daily during the treatment phase, and the last two weeks of theobservation period. These records included a rating of the stool usingthe Bristol Stool Form scale (1=very hard, 7=liquid).

Example 9: Assessments of Autism and Related Symptoms

Autism Diagnostic Interview-Revised (ADI-R) is a 2-hour structuredinterview and is one of the primary tools used for clinical diagnosis ofautism and autism spectrum disorders. It is not designed to be a measureof autism severity, but higher scores are generally consistent with moresevere symptoms. The ADI-R was be used to verify the diagnosis of ASDfor admission into the study.

Parent Global Impressions-III is introduced here as an expanded versionof the PGI-R. See Adams et al., Effect of a Vitamin/Mineral Supplementon Children with Autism, BMC Pediatrics, 11:111 (2011). The PGI-IIIevaluates changes in 17 areas (see FIG. 13), and overall, using a7-point scale ranging from “much worse” to “much better”. An “AverageChange” is computed by computing the average in all 18 scores of thePGI-2-Final. This tool was chosen because it was found that it is morereliable to ask parents directly about observed changes than to havethem estimate symptom severity at beginning and end and then compute adifference. Also, the use of a 7-point scale to detect changes seems toyield a high sensitivity to changes.

Childhood Autism Rating Scale (CARS) is a 15-item scale that can be usedto both diagnose autism and ASD and to assess the overall severity ofsymptoms. The CARS assessment was done subsequent to the ADIR assessmentby the same evaluator.

Aberrant Behavior Checklist (ABC) assesses problem behaviors in fiveareas common in children with ASD, including irritability, lethargy,stereotypy, hyperactivity, and inappropriate speech.

Social Responsiveness Scale (SRS) is a 65-item scale that assessessocial impairments, a core issue in autism, including social awareness,social information processing, capacity for reciprocal socialcommunication, social anxiety/avoidance, and autistic preoccupations andtraits. See Constantino et al., Validation of a brief quantitativemeasure of autistic traits: comparison of the social responsivenessscalewith the autism diagnostic interview-revised. J Autism Dev Disord. 2003August; 33(4):427-33.

Vineland Adaptive Behavior Scale II (VABS-II) is a measure of thefunctioning level in four different domains: Communication, Daily LivingSkills, Socialization, and Motor Skills, and 11 sub-domains. The rawscores were converted into an age equivalent score. It complements theABC, which assesses problem behaviors. See Sara et al., VinelandAdaptive Behavior Scales, Second Edition (Vineland™-II), PearsonPublishing, 2005.

The GSRS and PGI-R3 were assessed on days 0, 7, 14, 21, 28, 35, 42, 56,74, and 130. The Stool Record was assessed every day during thetreatment. The CARS, ABC, and SRS were assessed at baseline, at the endof treatment, and at the end of the observation period. The VABS-II wasassessed at baseline and at the end of the observation period only,because it is lengthy and we believed it is less sensitive to short timeperiods since it assesses changes in specific adaptive skills. The CARSwas assessed by a professional evaluator, and the GSRS, PGI-R2, ABC,SRS, and VABS-II were assessed by parents.

Example 10: Initial Observations

Gi Symptoms:

During the 2 weeks of vancomycin and then 8 weeks of beneficialbacteria, there was a rapid improvement in GI symptoms in most children.At the end of treatment there was an 82% reduction in average scores onthe Gastrointestinal Symptom Rating Scale (GSRS) (FIG. 1 and FIG. 3). Asshown in FIG. 2 and FIG. 5, roughly equal decrease in all 4 GSRSsubscale areas (abdominal pain, indigestion, diarrhea, constipation).There was no change in the reflux subscale because none of the childrenhad a significant reflux problem. Sixteen of 18 children had a 70% orgreater reduction, 1 had a 30% reduction, and 1 exhibited no change.Similar results were obtained for both the rectal-administration groupand the oral-administration group.

Autism Symptoms:

By the end of the treatment phase, the parents rated their children'sautism symptoms on the Overall scale of the Parent Global Impressionsas: Much Better—4; Better—8; Slightly Better—5; Little/No change—1. Thelargest improvements were in GI, speech, sociability, receptivelanguage, cognition, irritability/mood, anxiety, and play skills (FIG.3). For the Childhood Autism Rating Scale (CARS) rated by ourexperienced evaluator, there was a 22% decrease in the CARS scores,p<0.001, which is consistent with the observations by the parents. Forthe Aberrant Behavior Checklist (ABC), there was a 27% reduction in thetotal score, p=0.001 (FIG. 4). Similar results were obtained for boththe rectal-administration group and the oral-administration group.

Post-Treatment:

Among the first 5 participants that completed the 8-week post-treatmentobservation period, after two months of receiving no treatment, onaverage no change in improvements of GI symptoms was observed (73%reduction in GSRS at end of treatment vs. start; 71% reduction after 8weeks of no treatment vs. start). With respect to post-treatment autismsymptoms, PGI-Scores continued to improve over those collected at theend of treatment, with medium to large improvements in 3 participantsand no detected change in 2 participants. (FIG. 7). With regard topost-treatment CARS scores, these 5 children had a 16% decrease in CARSscores at the end of treatment, and a 25% decrease compared to baselineat the end of the no-treatment (observation) period. So, there appearedto be a surprising continued improvement in symptoms even aftertreatment stopped.

These data demonstrates a 22% reduction in autism severity scoresassigned using the Childhood Autism Rating Scale (CARS) after only 10weeks of the combined therapy (FIG. 3). The degree of improvement on theCARS did not appear to correlate with age (FIG. 6). This suggests thatthe treatment is useful for both younger children and adults.Furthermore, the degree of improvement on the CARS did not correlatewith initial GSRS score (FIG. 8). This suggests that the treatment ishelpful to those with mild GI symptoms as well as those without GIsymptoms. In other words, the treatment appears to be effective toreduce autism symptoms regardless of the presence or absence of GIsymptoms. This observation is consistent with data reported in ourprevious study (Kang et al., PLOS One 8(7):e68322 (2013)), from which weconcluded that children with ASD had a low diversity of gut bacteriathat was independent of their gastrointestinal symptoms.

Example 11: Final Results and Analysis

Clinically, this study was broadly successful. First, all ASDparticipants completed the 18-week study. Second, GI symptoms, asassessed by the Gastrointestinal Symptom Rating Scale (GSRS),significantly improved for abdominal pain, indigestion, diarrhea, andconstipation, such that the average GSRS score dropped 82% from thebeginning to end of treatment and remained improved (77% decrease frombaseline) at 8 weeks after treatment stopped (two-tailed paired t-testt=−9.45, p<0.001, t=−7.64, p<0.001, respectively) (FIG. 9, panel a). Asteady and large degree of improvement in most areas of GSRS evaluationincluding abdominal pain, indigestion, diarrhea, and constipation (FIG.10, panel a) was observed. There was little change in reflux since nochildren had significant reflux at the start of the study. Notably, twoseemingly opposite GI symptoms—diarrhea and constipation—responded tothe MTT treatment effectively.

Similarly, the Daily Stool Record (DSR), showed significant decreases inthe number of days with abnormal or no stools, and those improvementsremained after 8 weeks of no treatment (Table 5, FIG. 10, panel b). TheDaily Stool Record (DSR) was collected and averaged it over two weeks inorder to assess changes in stool hardness/softness during the study.Overall, we observed a significant decrease in “% days of abnormalstool” that combines % days of hard, soft/liquid, and no stool, from 62%to 34% (p=0.001) during the 10-week MTT treatment (Table 5 and FIG. 10,panel b). The improvements remained stable for the following 8 weeksduring the observation period. In detail, both “% days of hard stools”(type 1 or 2) and “% days of soft/liquid stools” (type 6 or 7)significantly decreased during the 10-week MTT treatment, but thedecrease in “% days of no stool” was not significant. (Table 5).

TABLE 5 Percent days of no stool, stool hardness and softness based onthe daily stool record (DSR) and the Bristol Stool Form Scale. 8 weeksTreatment after p- Baseline end p-value treatment value No stool 33% 26%0.27 26% 0.38 Hard stool 19%  6% 0.04  3% 0.01 (type 1 or 2) Soft/liquidstool 10%  2% 0.05  3% 0.11 (type 6 or 7) Abnormal stool 62% 34% 0.000732% 0.001 (in total of hard, soft/liquid/, no stool)

Third, there were only temporary adverse effects (primarily mild tomoderate hyperactivity and tantrums/aggression) from vancomycintreatment (Table 4), but no major changes in blood chemistry orlong-term adverse effects.

Beyond these GI improvements, ASD-related behavior also improvedfollowing MTT. First, the Parent Global Impressions (PGI-R) assessment,which evaluates 17 ASD-related symptoms, revealed significantimprovement during treatment and no reversion 8 weeks after treatmentended (FIG. 9, panel b). Further, a significant negative correlationbetween GSRS and PGI-R (Spearman correlation test showed r=−0.59 andp<0.001, FIG. 11) suggests that GI symptoms impact ASD behaviors, andthat these can be altered via MTT. By the end of the MTT treatment atweek 10, the parents rated the change in their children's autismsymptoms using the PGI-R, and the largest improvements were in the GIsubscore among 17 subscales and “Overall autism/related symptoms” of thePGI-R (FIG. 12). Specifically, the overall scale of PGI-R was rated asMuch Better: n=4 (22%); Better: n=8 (44%); Slightly Better: n=5 (28%);Little/No change: n=1 (6%). The improvement in the other subscales isshown in FIG. 12.

Second, the Childhood Autism Rating Scale (CARS), which rates core ASDsymptoms, decreased by 22% from beginning to end of treatment and 24%(relative to baseline) after 8 weeks of no treatment (p<0.001, FIG. 9,panel c).

Third, ASD-afflicted children saw improvement in their scores in theSocial Responsiveness Scale (SRS), which assesses social skill deficits(FIG. 9, panel d), and the Aberrant Behavior Checklist (ABC), whichevaluates irritability, hyperactivity, lethargy, stereotypy, andaberrant speech (FIG. 9, panel e). FIG. 10, panel c also shows a moredetailed breakdown of ABC analysis to assess treatment effects onbehaviors common in children with ASD: irritability, lethargy,stereotypy, hyperactivity, and inappropriate speech. In all fivesubscales, a significant reduction at the end of treatment was observed.

Fourth, the Vineland Adaptive Behavior Scale II (VABS-II) scoring foundthat the average developmental age increased by 1.4 years (p<0.001,VABS-II) and across all sub-domain areas (FIG. 13) during MTT; thoughthe final VABS-II score was still lower than their chronological age.VABS-II is a measure of the functioning level in four different domains:Communication, Daily Living Skills, Socialization, and Motor Skills,based on 11 sub-domains. Among 11 subscales, Fine and Gross Motor skillswere excluded, since these two subscales for the Vineland are onlycalculated up to 6.8 years and most children with ASD improved near tothe limit of the scale. The other 9 subscales and their average werecompared between the baseline and at the end of the study. The MTTtreatment resulted in a significant increase in average developmentalage, from 5.4 years at baseline to 6.8 years at the end of the study(p<0.001). A gain of 1.4 years within 18 weeks of the study is asubstantial increase, but they still remained below their chronologicalage of 10.9 years. significant improvements were also observed in all 9subscale areas with the largest gains in Interpersonal Skills (2.2years), Personal Living Skills (1.8 years), and Coping Skills (1.7years) (FIG. 13). It is notable that the major impairments in ASD,namely Receptive language, Expressive language, and Interpersonalskills, were among the lowest initial scores, with initial developmentalages of 3.1 years, 4.5 years, and 2.9 years, respectively; all threeareas had substantial improvements of 1.3, 1.1, and 2.2 years,respectively.

Finally, the MTT appears to be beneficial across both younger and olderindividuals (no significant correlations between age and GSRS or CARSimprovement) and whether the initial MTT does was received orally orrectally. Under our sample size, no difference was observed in efficacyof treatment or clinical outcomes whether MTT was initially administeredrectally or orally.

Together these findings show that MTT is safe and well-tolerated acrossan age-diverse cohort of 18 ASD-afflicted children. MTT is alsoeffective as it led to significant improvements in both GI- andbehavior-related symptoms that were sustained at least 8 weeks aftertreatment.

1-20. (canceled)
 21. A method for treating an autism spectrum disorder(ASD) in a subject in need thereof, the method comprising administeringto the subject (i) a pharmaceutical composition comprising asubstantially entire microbiota of a stool sample of a human donor; and(ii) a bacterial isolate comprising bacteria of the genus Lactobacillus.22. The method of claim 21, wherein the pharmaceutical compositioncomprises the bacterial isolate.
 23. The method of claim 21, wherein thetreating comprises a reduction in severity of a symptom of the ASDselected from the group consisting of: irritability, agitation,lethargy, social withdrawal, stereotypic behavior, hyperactivity,noncompliance, inappropriate speech, and a combination thereof.
 24. Themethod of claim 23, wherein the subject exhibits at least a 10%reduction in severity of the symptom as a result of the treating. 25.The method of claim 24, wherein the reduction in severity of the symptomis based on an assessment system selected from the group consisting of:Childhood Autism Rating Scale (CARS), Childhood Autism Rating Scale2-Standard Form (CARS2-ST), Childhood Autism Rating Scale 2-HighFunctioning (CARS2-HF), and a combination thereof.
 26. The method ofclaim 21, wherein the subject is a human below the age of
 18. 27. Themethod of claim 21, wherein the subject is a human adult.
 28. The methodof claim 21, wherein at least one of the pharmaceutical composition andthe bacterial isolate is administered orally.
 29. The method of claim21, wherein the method further comprises pretreating the subject with anantibiotic.
 30. The method of claim 21, wherein the subject exhibits oneor more gastrointestinal symptoms selected from the group consisting of:abdominal pain, reflux, indigestion, irritable bowel syndrome, chronicpersistent diarrhoea, diarrhoea, flatulence, constipation, andalternating constipation/diarrhoea.
 31. The method of claim 21, whereinthe ASD is selected from the group consisting of: autistic disorder,pervasive developmental disorder not otherwise specified (PDD-NOS), andAsperger syndrome.
 32. The method of claim 21, wherein thepharmaceutical composition and the bacterial isolate are formulatedtogether in a geltab, pill, microcapsule, capsule, or tablet.
 33. Amethod for treating an autism spectrum disorder (ASD) in a subject inneed thereof, the method comprising administering to the subject apharmaceutical composition comprising (i) a microbiota extracted from astool sample of a human donor; and (ii) cultured bacteria comprisingbacteria of the genus Lactobacillus.
 34. The method of claim 33, whereinthe treating comprises a reduction in severity of a symptom of the ASDselected from the group consisting of: irritability, agitation,lethargy, social withdrawal, stereotypic behavior, hyperactivity,noncompliance, inappropriate speech, and a combination thereof.
 35. Themethod of claim 34, wherein the reduction in the severity of the symptomis based on an assessment system selected from the group consisting of:Childhood Autism Rating Scale (CARS), Childhood Autism Rating Scale2-Standard Form (CARS2-ST), Childhood Autism Rating Scale 2-HighFunctioning (CARS2-HF), and a combination thereof.
 36. The method ofclaim 33, wherein the subject is a human below the age of
 18. 37. Themethod of claim 33, wherein the subject is a human adult.
 38. The methodof claim 33, wherein the pharmaceutical composition is administeredorally.
 39. The method of claim 33, wherein the method further comprisespretreating the subject with an antibiotic.
 40. The method of claim 33,wherein the subject exhibits one or more gastrointestinal symptomsselected from the group consisting of: abdominal pain, reflux,indigestion, irritable bowel syndrome, chronic persistent diarrhoea,diarrhoea, flatulence, constipation, and alternatingconstipation/diarrhoea.
 41. The method of claim 33, wherein the ASD isselected from the group consisting of: autistic disorder, pervasivedevelopmental disorder not otherwise specified (PDD-NOS), and Aspergersyndrome.
 42. The method of claim 33, wherein the pharmaceuticalcomposition and the bacterial isolate are formulated together in ageltab, pill, microcapsule, capsule, or tablet.